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Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, <t>CD9,</t> and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.

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1) Product Images from "Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice"

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22041672

Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, CD9, and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.

Figure Legend Snippet: Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, CD9, and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.

Techniques Used: Injection, Control, Staining, Isolation, Immunofluorescence, Negative Control, Light Microscopy, Microscopy

CP-treated immature mice showed a significant decrease in the number of subpopulations of spermatogenic cells compared to control: Cyclophosphamide (CP)- or PBS-treated mice (control, CT) were i.p injected as described in . Ten days post-treatment, testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic cells that express α-6-INTEGRIN, VASA, CD9, GFR-α, and c-KIT, or the meiotic cells that express the markers BOULE and CREM and the meiotic/post-meiotic cells that express the marker ACROSIN were identified by immunofluorescence staining using specific primary antibodies for each cell marker and the secondary antibody Cy3 (red color). DAPI (blue color) stained the nucleus of the cells (( A , B ), respectively). The identified premeiotic, meiotic, and meiotic/post-meiotic cells were counted, and their number/testis was evaluated (( C , D ), respectively). Arrows indicate the stained cells. ***— p < 0.001.

Figure Legend Snippet: CP-treated immature mice showed a significant decrease in the number of subpopulations of spermatogenic cells compared to control: Cyclophosphamide (CP)- or PBS-treated mice (control, CT) were i.p injected as described in . Ten days post-treatment, testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic cells that express α-6-INTEGRIN, VASA, CD9, GFR-α, and c-KIT, or the meiotic cells that express the markers BOULE and CREM and the meiotic/post-meiotic cells that express the marker ACROSIN were identified by immunofluorescence staining using specific primary antibodies for each cell marker and the secondary antibody Cy3 (red color). DAPI (blue color) stained the nucleus of the cells (( A , B ), respectively). The identified premeiotic, meiotic, and meiotic/post-meiotic cells were counted, and their number/testis was evaluated (( C , D ), respectively). Arrows indicate the stained cells. ***— p < 0.001.

Techniques Used: Control, Injection, Isolation, Marker, Immunofluorescence, Staining

Effect of hormones (FSH and testosterone) and cytokines (IL-1α and TNFα) on the proliferation and differentiation of spermatogonial cells isolated from CP-treated immature mice cultured in vitro in MCS.

Figure Legend Snippet: Effect of hormones (FSH and testosterone) and cytokines (IL-1α and TNFα) on the proliferation and differentiation of spermatogonial cells isolated from CP-treated immature mice cultured in vitro in MCS.

Techniques Used: Isolation, Cell Culture, In Vitro

Isolated cells from seminiferous tubules of CP-treated immature mice developed colonies in vitro in methylcellulose culture system (MCS): Isolated cells from seminiferous tubules of CP-treated immature mice, ten days after the last injection were cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF, LIF, FGF, EGF) as described in materials and methods section in the absence or presence of IL-1α, TNF-α, FSH, testosterone (T), or both IL-1α + T, TNF-α + T, FSH + T. Developed colonies after 4–5 weeks of culture are presented ( A ). The developed cells in the different treatments were positively stained for premeiotic markers (VASA, CD9, α-6-integrin, C-KIT), meiotic markers (Boule, Crem) and meiotic/post-meiotic marker (Acrosin) as examined by immunofluorescence staining using specific primary antibodies for each cell type and the secondary antibody Cy3 (red color) and DAPI (blue color) that stained the nucleus of the cells ( B ). Scale bare: 100 μm.

Figure Legend Snippet: Isolated cells from seminiferous tubules of CP-treated immature mice developed colonies in vitro in methylcellulose culture system (MCS): Isolated cells from seminiferous tubules of CP-treated immature mice, ten days after the last injection were cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF, LIF, FGF, EGF) as described in materials and methods section in the absence or presence of IL-1α, TNF-α, FSH, testosterone (T), or both IL-1α + T, TNF-α + T, FSH + T. Developed colonies after 4–5 weeks of culture are presented ( A ). The developed cells in the different treatments were positively stained for premeiotic markers (VASA, CD9, α-6-integrin, C-KIT), meiotic markers (Boule, Crem) and meiotic/post-meiotic marker (Acrosin) as examined by immunofluorescence staining using specific primary antibodies for each cell type and the secondary antibody Cy3 (red color) and DAPI (blue color) that stained the nucleus of the cells ( B ). Scale bare: 100 μm.

Techniques Used: Isolation, In Vitro, Injection, Cell Culture, Staining, Marker, Immunofluorescence

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Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Cyclophosphamide significantly decreased the testicular weight and seminiferous tubule normal histology, VASA cells GFR-α-1, α-6-Integrin, CD9, and C-KIT cells counts in the tubules of immature mice: cyclophosphamide (CP) was intraperitoneally injected (i.p; 100 mg/kg in 100 uL; see methodology section) (CP) or PBS (control, CT; 100 uL). One to 5 weeks after the last injection, mice were sacrificed, and testes were removed, weighed, and fixed in Bouin’s solution for histological evaluation. Changes in the testes weight following CP treatment (CP) compared to control (Control) is presented ( A ). The histology of the seminiferous tubules was examined by hematoxylin-eosin staining ( B ) and a summary of seminiferous tubule damage after 1–5 weeks post CP (CP) treatment compared to the CT is presented ( C ). Ten days post-treatment, the histology of the seminiferous tubules was evaluated by H&E staining ( D ), testes were weighed ( E ), and the total number of cells isolated from the seminiferous tubules were counted ( F ). The presence of VASA-, GFR-α-1-, α-6-Integrin-, CD9-, and C-KIT-positive stained cells in the seminiferous tubules of CT and CP-treated immature mice ( G – K ) was examined by immunofluorescence staining (IF) using specific primary antibodies and Cy3 or Alexa-flour 488 with the relevant secondary antibodies (VASA, α-6-Integrin, CD9, and C-KIT red staining and GFR-α-1 green staining). DAPI (blue color) stained the nucleus of the cells. Arrows show the location of stained cells in the testicular tissues. As a negative control (NC), we stained the tissues only with the secondary antibodies (NC for α-6-Integrin, CD9 and C-KIT were similar and therefore, we present only NC for α-6-Integrin). ( B )—X20 light microscope magnification (100 µm scale). ( D )—X40 light microscope magnification (100 µm scale). ( G – K )—X40 fluorescent microscope magnification (100 µm scale). **— p < 0.01 and ***— p < 0.001.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: Monoclonal mouse anti-mouse Vimentin (Novus, Littleton, CO, USA; 1:500), and polyclonal goat anti-mouse α- sma (Abcam, 1:250), Polyclonal goat anti-mouse Integrin α6 (Santa Cruz, CA, USA; 1:40), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz; 1:100), monoclonal mouse anti-mouse GFR-α-1 (Santa Cruz, sc-271546; 1:50), monoclonal mouse anti-mouse α-6-INTEGRIN (Santa Cruz, 1:50), monoclonal mouse anti-mouse CD9 (Santa Cruz, 1:50), and monoclonal mouse anti-mouse C-KIT (Santa Cruz, 1:50), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Injection, Control, Staining, Isolation, Immunofluorescence, Negative Control, Light Microscopy, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: CP-treated immature mice showed a significant decrease in the number of subpopulations of spermatogenic cells compared to control: Cyclophosphamide (CP)- or PBS-treated mice (control, CT) were i.p injected as described in . Ten days post-treatment, testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic cells that express α-6-INTEGRIN, VASA, CD9, GFR-α, and c-KIT, or the meiotic cells that express the markers BOULE and CREM and the meiotic/post-meiotic cells that express the marker ACROSIN were identified by immunofluorescence staining using specific primary antibodies for each cell marker and the secondary antibody Cy3 (red color). DAPI (blue color) stained the nucleus of the cells (( A , B ), respectively). The identified premeiotic, meiotic, and meiotic/post-meiotic cells were counted, and their number/testis was evaluated (( C , D ), respectively). Arrows indicate the stained cells. ***— p < 0.001.

Techniques: Control, Injection, Isolation, Marker, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Effect of hormones (FSH and testosterone) and cytokines (IL-1α and TNFα) on the proliferation and differentiation of spermatogonial cells isolated from CP-treated immature mice cultured in vitro in MCS.

Techniques: Isolation, Cell Culture, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice

doi: 10.3390/ijms22041672

Figure Lengend Snippet: Isolated cells from seminiferous tubules of CP-treated immature mice developed colonies in vitro in methylcellulose culture system (MCS): Isolated cells from seminiferous tubules of CP-treated immature mice, ten days after the last injection were cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF, LIF, FGF, EGF) as described in materials and methods section in the absence or presence of IL-1α, TNF-α, FSH, testosterone (T), or both IL-1α + T, TNF-α + T, FSH + T. Developed colonies after 4–5 weeks of culture are presented ( A ). The developed cells in the different treatments were positively stained for premeiotic markers (VASA, CD9, α-6-integrin, C-KIT), meiotic markers (Boule, Crem) and meiotic/post-meiotic marker (Acrosin) as examined by immunofluorescence staining using specific primary antibodies for each cell type and the secondary antibody Cy3 (red color) and DAPI (blue color) that stained the nucleus of the cells ( B ). Scale bare: 100 μm.

Techniques: Isolation, In Vitro, Injection, Cell Culture, Staining, Marker, Immunofluorescence

Effect of 10-day post busulfan (BU) treatment on spermatogonial cell counts and percentages, as examined by fluorescence-activated cell sorter (FACS) analysis, as follows: BU or DMASO (control, CT) were i.p injected, as described in . Ten days after the injection, the testes were removed, seminiferous tubules were separated, and the cells were enzymatically isolated from the seminiferous tubules. Spermatogonial cells with the membrane markers: alpha-6-INTEGRIN (alpha-6-INT), c-KIT, G-CSF-R, and THY1 were identified by FACS using specific antibodies for each marker ( A ). Spermatogenic cells were identified by immunofluorescence staining for specific markers [premeiotic (CD9, VASA, and SALL4), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN)] ( B ). The above identified spermatogonial cells were counted and calculated per testicle ( C ), and their percentage was evaluated ( D ). ** p < 0.01 and *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice

doi: 10.3390/ijms19123804

Figure Lengend Snippet: Effect of 10-day post busulfan (BU) treatment on spermatogonial cell counts and percentages, as examined by fluorescence-activated cell sorter (FACS) analysis, as follows: BU or DMASO (control, CT) were i.p injected, as described in . Ten days after the injection, the testes were removed, seminiferous tubules were separated, and the cells were enzymatically isolated from the seminiferous tubules. Spermatogonial cells with the membrane markers: alpha-6-INTEGRIN (alpha-6-INT), c-KIT, G-CSF-R, and THY1 were identified by FACS using specific antibodies for each marker ( A ). Spermatogenic cells were identified by immunofluorescence staining for specific markers [premeiotic (CD9, VASA, and SALL4), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN)] ( B ). The above identified spermatogonial cells were counted and calculated per testicle ( C ), and their percentage was evaluated ( D ). ** p < 0.01 and *** p < 0.001.

Article Snippet: Following the removal of the blocking buffer, the first antibodies were added, as follows: polyclonal rabbit anti-mouse SALL4 (Abcam, Cambridge, UK; 1:400), polyclonal rabbit anti-mouse VASA (Santa Cruz; 1:100), polyclonal rabbit anti-mouse CD9 (Santa Cruz, CA, USA; 1:100), polyclonal rabbit anti-mouse BOULE (Santa Cruz; 1:50), polyclonal rabbit anti-mouse CREM-1 (Santa Cruz; 1:50), and polyclonal rabbit anti-mouse ACROSIN (Santa Cruz; 1:200).

Techniques: Fluorescence, Control, Injection, Isolation, Membrane, Marker, Immunofluorescence, Staining

Effect of 10-day post busulfan (BU) treatment on spermatogenic cell counts, percentages and expression: BU or DMASO (control, CT) were i.p injected, as described in . Ten days after the injection, the testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic (CD9, VASA, and SALL4), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN) cells were identified by immunofluorescence staining using the antibodies specific for each marker ( B). The above identified spermatogenic cells were counted and calculated per testicle ( A ), and their percentage was evaluated ( B ). Their expression was analyzed by qPCR analysis (fold of increase compared to control) ( C ). ×40 light microscope magnification (100 µm scale). ** p < 0.01 and *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice

doi: 10.3390/ijms19123804

Figure Lengend Snippet: Effect of 10-day post busulfan (BU) treatment on spermatogenic cell counts, percentages and expression: BU or DMASO (control, CT) were i.p injected, as described in . Ten days after the injection, the testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated from the seminiferous tubules. The premeiotic (CD9, VASA, and SALL4), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN) cells were identified by immunofluorescence staining using the antibodies specific for each marker ( B). The above identified spermatogenic cells were counted and calculated per testicle ( A ), and their percentage was evaluated ( B ). Their expression was analyzed by qPCR analysis (fold of increase compared to control) ( C ). ×40 light microscope magnification (100 µm scale). ** p < 0.01 and *** p < 0.001.

Techniques: Expressing, Control, Injection, Isolation, Immunofluorescence, Staining, Marker, Light Microscopy

Development of spermatogenesis in vitro from BU-treated immature mice: Ten days after BU injection (see ), the testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated and cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF (glial cell line derived nerve growth factor), LIF (leukemia inhibitory factor), FGF (fibroblast growth factor), and EGF (epidermal growth factor)) (see Methodology). In some wells, we also added TNF-α (20 pg/mL), FSH (7.5 IU/mL), or testicular homogenates from immature mice (60 µg/mL) or testicular homogenates from GFP-adult mice (60 µg/mL). Every 10–14 days, we added new media containing the same composition of factors that was added in the beginning of the culture. After 4–6 weeks, the developed colonies and cells ( A ) were collected, and the cells were fixed using cold methanol and were stained using immunofluorescence staining, using specific antibodies for markers of the premeiotic (CD9 and VASA), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN) cells. The effect of FSH and TNF-alpha ( B ) testicular homogenates from immature ( C ) or adult ( D ) mice on the development of spermatogenic cells in vitro compared to before the culture or the control (CT; after culture in the presence of growth factors) were examined. In addition, the development of the sperm-like cells in the cultures was examined according to the DAPI staining of sperm head morphology [ E ; E1—positive control of a sperm head (H); E2—a single sperm head developed in the culture; E3—a group of sperm heads developed in the culture] or periodic acid–Schiff (PAS) staining [ F ; F1—a group of sperm-like cells with heads (H) and tails; F2—a few sperm-like cells with complete morphology of head (H), neck (N), and tails (T)]. ×40 light microscope magnification (100 µm scale). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice

doi: 10.3390/ijms19123804

Figure Lengend Snippet: Development of spermatogenesis in vitro from BU-treated immature mice: Ten days after BU injection (see ), the testes were removed, seminiferous tubules were separated, and cells were enzymatically isolated and cultured in a methylcellulose culture system (MCS). The MCS was composed of 42% methylcellulose, KSR (10%), StemPro, and growth factors (GDNF (glial cell line derived nerve growth factor), LIF (leukemia inhibitory factor), FGF (fibroblast growth factor), and EGF (epidermal growth factor)) (see Methodology). In some wells, we also added TNF-α (20 pg/mL), FSH (7.5 IU/mL), or testicular homogenates from immature mice (60 µg/mL) or testicular homogenates from GFP-adult mice (60 µg/mL). Every 10–14 days, we added new media containing the same composition of factors that was added in the beginning of the culture. After 4–6 weeks, the developed colonies and cells ( A ) were collected, and the cells were fixed using cold methanol and were stained using immunofluorescence staining, using specific antibodies for markers of the premeiotic (CD9 and VASA), meiotic (CREM, BOULE, and ACROSIN), and postmeiotic (ACROSIN) cells. The effect of FSH and TNF-alpha ( B ) testicular homogenates from immature ( C ) or adult ( D ) mice on the development of spermatogenic cells in vitro compared to before the culture or the control (CT; after culture in the presence of growth factors) were examined. In addition, the development of the sperm-like cells in the cultures was examined according to the DAPI staining of sperm head morphology [ E ; E1—positive control of a sperm head (H); E2—a single sperm head developed in the culture; E3—a group of sperm heads developed in the culture] or periodic acid–Schiff (PAS) staining [ F ; F1—a group of sperm-like cells with heads (H) and tails; F2—a few sperm-like cells with complete morphology of head (H), neck (N), and tails (T)]. ×40 light microscope magnification (100 µm scale). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Techniques: In Vitro, Injection, Isolation, Cell Culture, Derivative Assay, Staining, Immunofluorescence, Control, Positive Control, Light Microscopy

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