Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Antibody Details

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Concentration Assay, Purification, Marker, Sequencing

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Non-neuronal components of the OE can be identified with various antibodies. A. DAB staining with the antibody to the transcription factor Hes1 labels nuclei of the supporting cells. B. The antibody to transcription factor Sox9 labels duct/gland cell nuclei only. C. Antibodies to K18 label both sustentacular cells as well duct/gland cells. D. Antibodies to E-Cadherin stain with a similar pattern to K18. E,F,G. Double immunofluorescent labeling of a sharp transitional zone between olfactory and respiratory epithelium with antibodies to Beta-tubulin IV and PGP9.5. Beta-tubulin IV densely labels the apical portion and cilia of the respiratory cells. Within the OE, PGP9.5 labels the cell body, axons and dendrites of the olfactory neurons. Several ciliated cells within the respiratory epithelium are labeled with both antibodies (arrows) and are clearly non-neuronal. Bg = bowman’s glands, arrowheads = basement membrane, scalebar = 25µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Labeling

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Multiple antibodies can be used to identify subpopulations of olfactory neurons (ONs). A. DAB staining with OMP antibodies label mature ONs that tend to reside in the more apical portion of the epithelium. B. Antibodies to Tuj-1 label both mature and immature ONs with dense labeling of the dendrites and axons. C. The antibody to Golf labels mature and immature ONs with at a higher density in the dendrites and cell bodies. Arrows = dendritic knobs. D,E,F. Double immunofluorscent labeling of the epithelium with antibodies to PGP9.5 and OMP demonstrates the relative numbers of mature vs. immature ONs. Normal olfactory epithelium contains abundant OMP(+)/PGP9.5(+) mature neurons and OMP(−)/PGP9.5(+) immature neurons (arrows) suggesting a dynamic neuroepithelium. G,H,I. With double immunofluorescent labeling of the epithelium using HBC marker K5 and ON marker PGP9.5, cells are identified just above the HBCs that are absent of staining for either antibody (arrows). These cells may be GBCs or early neuroprogenitor cells. Asterisk identifies olfactory nerve fasicles extending out from the basal layer of the epithelium. Arrowheads = basement membrane, scale bar = 25µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Labeling, Marker

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Transcription factor antibodies allow us to identify a subset of globose basal cells (GBCs). Immunofluorescent staining with Mash1 antibodies label clusters of cells in the OE (B,E,H,K). A,B,C. Mash1(+) cells (arrows) are not neurons as shown by lack of co-labeling with the anti-neuronal antibody Tuj-1. D,E,F. Mash1(+) cells are not HBCs as shown by absence of double staining with p63 antibodies. An arrow identifies a Mash1 stained cell sitting just above a p63 stained HBCs in a position typically occupied by GBCs. G,H,I. The absence of p27 double labeling with Mash1 (arrows) suggests that these cells are not quiescent. J,K,L. Many Mash1(+) cells (arrows) are mitotically active as shown by double labeling with Ki67 antibodies. M,N,O. Notch1 antibodies also label cells within the GBC compartment (arrows). Arrowheads = basement membrane, scale bar = 25µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Labeling, Double Staining

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Staining Characteristics by Cell Type

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Activity Assay

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Esthesioneuroblastoma can be characterized using antibody reagents similar to that used for OE. A. DAB stained section of a portion of an esthesioneuroblastoma taken from a 44 year-old female using the neuron-specific antibody Tuj-1 labels the majority of the tumor as expected. B. However, these cells are not mature ONs based on the absence of OMP staining. C. The sustentacular and gland/duct cell specific K18 antibody labels cords of cell separating the tumor into nests. (Scale bar = 250µ for A, B, and C.) D. The tumor does not stain with antibodies to K5, p63 (E), and Beta-Tubulin IV (F). Only cells of the epithelial surface overlying the tumor stain with antibodies to K5, p63, and Beta-Tubulin IV indicating a respiratory-like epithelium. (Scale bar = 50µ for D, E, and F.) G. DAB staining using the Golf antibody also labels a large portion of the tumor in a pattern similar to Tuj-1 supporting an olfactory epithelial origin of the tumor. An arrow indicates extension of the tumor cells into the overlying epithelium (scale bar = 50µ). H. Immunofluorescent staining using antibodies to Mash1 also stains a large portion of the tumor in a pattern similar to Tuj-1 and Golf and corroborates previous mRNA findings. Arrows indicate areas of tumor extension into the overlying epithelium (scale bar = 100µ). I. DAB staining with Pax6 antibodies labels a large portion of the tumor and the overlying epithelium (scale bar = 50µ). Arrowheads = basement membrane.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: Sox9(+)/Tuj-1(+) tumor cells do not follow the usual pattern of staining of olfactory epithelial gland cells. A,B,C. The separation of the tumor into two populations is confirmed using K18 and Tuj-1 antibodies. Similar to the two cell populations demonstrated with Sox2 and Sox9 antibodies, immunofluorescent labeling of cell cytoplasm and processes using K18 (arrows), does not co-label with the neuron-specific Tuj-1 antibody. D,E,F. E-Cad has a similar staining pattern as K18 in the epithelium labeling both gland and supporting cells, however within the tumor K18 is restricted to the Sox2(+)/Sox9(−)/Tuj-1(−) population while E-Cad labels the same NCAM(−) non-neuronal population (arrows) as well as the NCAM(+) neuronal population. Scale bar = 25µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Labeling

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: The esthesioneuroblastoma can be divided into two distinct populations using a combination of transcription factor and structural antibodies. A,B,C. Double immunofluorescent staining with Sox2 (arrows) and Sox9 antibodies show a mutually exclusive staining pattern within the tumor. Sox2(+) nuclei seem to occur at the periphery of the Sox9(+) cells and within the overlying epithelium (scale bar = 25µ). D,E,F. The staining of the tumor with Sox9 and K18 is also mutually exclusive and reveals a similar pattern to Sox9/Sox2 double staining (scale bar = 50µ). G,H,I. The Sox9 antibody labels Bowman’s duct/gland cell nuclei of the OE, but within the tumor it co-labels with Tuj-1(+) cells. The arrow indicates a neuronal process. Scale bar = 25µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Double Staining

Journal: The Laryngoscope

Article Title: Immunohistochemical characterization of human olfactory tissue

doi: 10.1002/lary.21856

Figure Lengend Snippet: A. The antibody to K17 labels only a portion of the tumor as shown with immunofluorescent staining and DAB at a lower magnification in the inset (inset scale bar = 100µ). B,C. Using double immunofluorescent staining with Tuj-1 a small number of cells are co-labeled with both antibodies (arrowheads), but most K17(+) cells seem to follow a pattern of staining similar to K18/Sox2 antibodies. White scale bar = 50µ.

Article Snippet: Some sections stained with DAB were counterstained with hematoxylin. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Species Concentration Company Catalogue # Antigen Beta-Tubulin IV Mouse 1:10000 Sigma T7941 c-terminal synthetic peptide beta-tubulin IV K17 Rabbit 1:1500 Abcam ab53707 c-terminal synthetic peptide human keratin 17 K18 Mouse 1:3000 Invitrogen 18–0158z human keratin 18 (45 kDa) polypeptide K18 Rabbit 1:9000 Abcam 52948-100 c-terminal synthetic peptide human keratin 18 K5/6 Mouse 1:150 Chemicon MAB1620 purified human keratin 5 E-Cad Mouse 1:300 Invitrogen 13–1700 human E-Cadherin EGFr Sheep 1:200 UBI 06–129 recombinate human EGF receptor G olf Rabbit 1:500 Santa Cruz sc-383 c-terminus of Gα s/olf of rat origin Hes1 Rabbit 1:2000 Generous gift from Dr. T. Sudo hes-1 protein Ki67 Rabbit 1:400 Epitomics 4203-1 c-terminal human Ki67 Mash1 Mouse 1:30 BD Biosciences 556604 recombinate full length rat Mash1 NCAM Rabbit 1:2500 Generous gift from Dr. Covault neural cell adhesion molecule Notch1 Rabbit 1:250 – 1:50 Cell Signaling D1E11 residues surrounding Pro2439 of a synthetic full-length peptide recognizing the transmembrane/intracellular region OMP Goat 1:12000 Wako 544–10001 rodent olfactory marker protein p27 Mouse 1:5000 BD Biosciences 610241 aa 1–197 mouse Kip1 p63 Mouse 1:1000 Santa Cruz sc-8431 aa 1–205 human p63 Pankeratin Rabbit 1:2000 DAKO Cytomation A0575 human keratins Pax6 Rabbit 1:6000 Chemicon AB5409 c-terminus synthetic human/mouse Pax6 PGP9.5 Rabbit 1:30000 Cedarlane RA95101 human brain protein gene product 9.5 Sox2 Goat 1:100 Santa Cruz sc-17320 c-terminus synthetic human Sox2 Sox9 Rabbit 1:2000 Millipore AB5535 sequence CCTTGAG synthetic peptide from human Sox9 Tuj-1 Mouse 1:300 Covance MMS-435P rat brain beta-tubulin III Open in a separate window Antibody Details WMs were incubated in 3% hydrogen peroxide for one hour and then dehydrated and re-hydrated through sequential dilutions of ethanol for 20 minutes each prior to staining.

Techniques: Staining, Labeling

Journal: Nature Communications

Article Title: YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells

doi: 10.1038/s41467-019-09404-6

Figure Lengend Snippet: YAP inhibition reduces Sox9 + progenitors in vitro. a Experimental design for b – e . b – d Flow cytometry of SOX9 expression in the control and verteporfin-treated differentiating progenitors and quantification of the proportion of SOX9 + cells from b and c at the end of stage 6 differentiation (stage 6, day 14). mCFP is an unstained control. Data represent mean ± SEM, ** p < 0.01 ( n = 3 biologically independent samples per group). e Proportion of Ki67 + in control or verteporfin-treated differentiating progenitors as assayed by flow cytometry at the end of stage 6 differentiation. Data represent mean ± SEM, *** p < 0.001 ( n = 3 biologically independent samples per group). f – h Flow cytometry analysis of SOX9 in non-targeting control and YAP shRNA-expressing pancreatic progenitors and quantification of SOX9 + cell proportion ( h ) at the end of stage 6 differentiation. Data represent mean ± SEM, *** p < 0.001 ( n = 3 biologically independent samples per group). i Experimental design for j – n . j , k in vivo GSIS of control and verteporfin-derived SC-β cell grafts as assayed by serum human insulin before and 30 min after a glucose injection 8 weeks post transplantation. l , m Immunofluorescence micrographs of grafts 12 weeks post transplantation of control and verteporfin-derived SC-β cell grafts stained for C-peptide and SOX9. Representative images and cropped blots (bottom panels) are shown. Asterisk (*) denotes nonspecific staining. Scale bar: 50 µm. n Quantification of SOX9 + of DAPI + cells within grafts 12 weeks after transplantation. Data represent mean (center line) ± min to max (whiskers) and lower and upper quartiles (bounds of box), *** p < 0.001, two-sided student’s t test ( n = 4 animals per group, values correspond to the average proportion of SOX9 + cells from four histological sections per animal)

Article Snippet: The antibodies used in the study were the following: rabbit anti-YAP (1:100, Cell Signaling Technology; 14074 S), mouse anti-YAP (1:100, Abnova; 89106308), rabbit anti-SOX9 (1:100, Cell Marque; AC-0284RUO), rat anti-C-peptide (1:200, Developmental Studies Hybridoma Bank; GN-ID4), mouse anti-NKX6.1 (1:100, Developmental Studies Hybridoma Bank; F55A12-supernatant), rabbit anti-Ki67 (1:100, Abcam; ab16667), sheep anti-NGN3 (1:50, R&D systems; AF3444), goat anti-PDX1 (1:100, R&D systems; AF2419), rabbit anti-CHGA (1:200, Novus Biologicals; NB120–15160), mouse anti-CHGA (1:200, Santa Cruz; sc-393941), mouse anti-glucagon (1:200, Abcam; ab82270), mouse anti-somatostatin (1:200, Santa Cruz; sc-55565), rabbit anti-cleaved Caspase-3 (1:100, Cell Signaling; 9661) and mouse anti-PCNA (1:100, Millipore; NA03).

Techniques: Inhibition, In Vitro, Flow Cytometry, Expressing, shRNA, In Vivo, Derivative Assay, Injection, Transplantation Assay, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: Antibodies

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques: Staining, Plasmid Preparation, Apoptosis Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: The Gdnf mRNA and GDNF and FGF2 protein levels in testes of 8-wk-old mice. (A) The Gdnf mRNA levels in WT, Het, and cKO mice relative to Sox9 levels. (B) The GDNF protein levels in WT, Het, and cKO mice relative to INHBB levels. (C) The FGF2 protein levels in WT, Het, and cKO mice relative to INHBB levels. ANOVA analysis with Tukey test. *P < 0.05, **P < 0.01.

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: Sertoli cell numbers, seminal vesicle weights, and the GDNF expression of 8-wk-old WT, Het, and cKO mice. (A) Sertoli cell nuclei (arrows) were immunostained with rabbit anti-mouse SOX9 antibody. (B) The numbers of Sertoli cells per tubule cross-sections of WT, Het, and cKO were not significantly different. (C) The seminal vesicle weights of WT, Het, and cKO mice were not significantly different. (D) The expression of GDNF in Sertoli cells of WT, Het, and cKO mice. Sections were immunostained with rabbit antiserum to GDNF and Alexa Fluor 546 donkey anti-rabbit antiserum (red) and with FITC-conjugated mouse monoclonal antibody to ACTA2 (green) to determine which cell types contained GDNF. GDNF was localized to Sertoli cells and PM cells in WT and Het mice but only in Sertoli cells in cKO mice. Nuclei were stained with DAPI. (Scale bar: 50 µm.)

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques: Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: PCR primers

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: The mRNA levels for markers of undifferentiated spermatogonia in testes of WT, Het, and cKO 1-wk-old mice relative to Sox9 levels. (A) Bcl6b. (B) Zbtb16. (C) Nanos2. ANOVA analysis with Tukey test. *P < 0.05, **P < 0.01.

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

doi: 10.1073/pnas.1517994113

Figure Lengend Snippet: The mRNA levels for markers of differentiated spermatogonia in testes of WT, Het, and cKO 1-wk-old mice relative to Sox9 levels. (A) Ngn3. (B) Kit. (C) Nanos3. (D) Spo11. (E) Dnmt3l. ANOVA analysis with Tukey test. *P < 0.05.

Article Snippet: Species Dilution Primary ACTA2 Sigma-Aldrich, A5228 (Clone 1A4) Mouse, monoclonal 1:200 ACTA2-FITC Sigma-Aldrich, F3777 (Clone 1A4) Mouse, monoclonal 1:200 dtTomato Clontech, 632496 Rabbit, polyclonal 1:500 GATA4 Santa Cruz, SC-1237 Goat, polyclonal 1:300 GDNF ( d -20) Santa Cruz, SC-328 Rabbit, polyclonal 1:200 GFP Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11122","term_id":"490966","term_text":"A11122"}} A11122 Rabbit, polyclonal 1:200 HSPA2 Eddy laboratory, Allen et al. PMID 3352605 Rabbit, polyclonal 1:1,000 KI67 Abcam, ab15580 Rabbit, polyclonal 1:200 KIT Abcam, ab65525 (Clone 2B8) Rat, monoclonal 1:100 SOX9 Cosmo Bio Co. Japan, KAL-KO608 Rabbit, polyclonal 1:200 ZBTB16 (PLZF, H-300) Santa Cruz, SC-22839 Rabbit, polyclonal 1:200 Secondary Alexa Fluor 488, Donkey anti-mouse IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 Donkey, polyclonal 1:200 Alexa Fluor 546, Donkey anti-rabbit IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103"}} A10040 Donkey, polyclonal 1:200 Alexa Fluro 647, Donkey anti-goat IgG Life Technologies, {"type":"entrez-nucleotide","attrs":{"text":"A11056","term_id":"489255","term_text":"A11056"}} A11056 Donkey, polyclonal 1:200 DAB staining VECTSTAIN Elite ABC Kit (Rabbit IgG) Vector Laboratories, PK-6106 ImmPACT DAB Peroxidase Substrate Vector Laboratories, SK-4105 Vybrant apoptosis Assay Kit 6 (V23200) Annexin V + Alexa Fluor 350 Life Technology PI Life Technology Open in a separate window Antibodies fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. S2. caption a7 ZBTB16 immunostaining of undifferentiated spermatogonia in testes from WT, Het, and cKO mice at 1, 2, 4, 8, and 12 wk. ( A ) No obvious differences are seen at 1 wk. ( B ) Fewer ZBTB16-positive cells were present in cKO testes compared to Het and WT at 2 wk. ( C – E ) ZBTB16-positive cells were rarely observed at 4 ( C ), 8 ( D ), and 12 ( E ) wk in cKO testes.

Techniques: