Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 2. Effects of deletions of hepatic nuclear factor 1 (HNF1) and CCAAT/enhancer binding protein (C/EBP) elements in the human ACAT2 gene on hepatocyte-specific expression. pC/EBP mutant, pHNF1 mutant, and pHNF1 and C/EBP mutant con- structs (A), carrying deletions for the putative C/EBP, the putative HNF1, and for both putative binding sites in the p1044 construct, respectively, were transiently transfected into HuH7 cells (B) and HepG2 cells (C). D: pHNF1 mutant p1305 construct, carrying a deletion for the putative HNF1 binding site in the full-length pro- moter construct, was transiently transfected into HepG2. pSV- -galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase activity. All transfections were per- formed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Expressing, Mutagenesis, Construct, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 3. Functionality of the HNF1 binding site in the human ACAT2 gene. Nuclear extracts, prepared from HuH7 cells, were incubated with 32P-labeled double-stranded probes with or without a mutation for the HNF1 binding site and resolved on a nondenatured (5%, w/v) acrylamide gel. For competition assays, a 100- fold molar excess of unlabeled probe was added to the binding reaction mixture, and for the supershift as- says, 2 g of HNF1 or HNF1 antibodies was added. Lanes 1 and 2, binding reaction between 10 and 20 g nuclear extracts, respectively, and 32P-labeled HNF1 probe. Lane 3, competition between labeled and unla- beled HNF1 probe. Lane 4, competition between labeled HNF1 probe and unlabeled mutated HNF1 probe. Lane 5, supershift reaction between labeled HNF1 probe and 2 g of HNF1 antibody. Lane 6, supershift re- action between labeled HNF1 probe and 2 g of HNF1 antibody. Lanes 7 and 8, binding reaction between 10 and 20 g nuclear extracts, respectively, and labeled mutated HNF1 probe. Lane 9, 10 g of BSA and la- beled HNF1 probe, serving as a negative control (Neg. Ctrl).

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Incubation, Labeling, Mutagenesis, Acrylamide Gel Assay, Negative Control

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 4. In vivo association of HNF1 and HNF1 with the human ACAT2 promoter in liver. Soluble chromatin was prepared from 200 mg of human liver and immunoprecipitated with specific anti- bodies against HNF1, HNF1, or IgG and amplified using real- time RT-PCR, conducted in triplicate. The IgG antibody was used as a baseline control and to compare the relative fold enrichment of the ACAT2 promoter by the specific DNA fragments. Before immu- noprecipitations, a small aliquot of chromatin was saved and used as an input control. ChIP, chromatin immunoprecipitation.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: In Vivo, Immunoprecipitation, Quantitative RT-PCR, Control, Chromatin Immunoprecipitation

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 5. Importance of the HNF1 binding site for hepatocyte-spe- cific expression. The p1044 construct (A) or the pHNF1 mu- tant construct (B) carrying a deletion for the putative HNF1 bind- ing site, and 0.1 or 0.5 g of HNF1 and/or HNF1 expression vector, was cotransfected into HuH7 cells. pSV--galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase activity. All transfections were performed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Binding Assay, Expressing, Construct, Plasmid Preparation, Control, Transfection, Luciferase, Activity Assay

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 6. Influence of HNF1 and HNF1 overexpression on hu- man ACAT2 promoter activity in different cells. The human ACAT2 full-length promoter construct (p1305), along with 0.1 or 0.2 g of HNF1 and/or HNF1 expression vector, was transiently cotransfected into HepG2 cells (A), into HuH7 cells (B), or into HEK293 cells (C). pSV--galactosidase (-Gal) control vector served as an internal control of transfection efficiency. The empty (pGL3) reporter vector served as a baseline control. Luciferase and -galactosidase activities were measured in cell lysates 48 h after transfection. Luciferase activity was corrected by -galactosidase ac- tivity. All transfections were performed in five replicates, and data represent means SEM.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Over Expression, Activity Assay, Construct, Expressing, Plasmid Preparation, Control, Transfection, Luciferase

Journal: Journal of Lipid Research

Article Title: Control of ACAT2 liver expression by HNF1

doi: 10.1194/jlr.m400450-jlr200

Figure Lengend Snippet: Fig. 7. Alignment between human and mouse 5 flanking regions of the ACAT2 gene. Shaded letters show base homology. aataaa, poly-A sequence of the IGFBP-6 gene; Cdx-2, caudal-related homeodomain protein-2; framed HNF1, sequence for the HNF1 responsible for the hepatocyte-specific expression of the ACAT2 gene in humans; (HNF1), cis-element for HNF1 present only in the mouse sequence. The longer underlined se- quence shows part of the exon sequence.

Article Snippet: antibody (catalog number Sc-6547X; Santa Cruz Biotechnology, Santa Cruz, CA) or 2 g of HNF1 antibody (catalog number Sc-22840X; Santa Cruz Biotechnology) was added to the binding reaction mixture.

Techniques: Sequencing, Expressing

Journal: Cell Death & Disease

Article Title: Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma

doi: 10.1038/s41419-023-06376-w

Figure Lengend Snippet: A Expression of CD276 in LUSC ( n = 502) and normal samples ( n = 51) based on TCGA. B The relationship between CD276 and CD8 + T cells (R = −0.188, P < 0.001) or CD4+ activated memory cells (R = −0.138, P < 0.001) in LUSC analyzed by Timer2.0. C Histochemistry score (H-score) of B7H3 on 10 samples of normal lung tissues, LUAD, LUSC, and SCLC shown by IHC staining. Scale bars = 200 μm. D H-score of B7H3 on 48 pairs of LUSC tissues and adjacent normal tissues shown by IHC. Scale bars = 100 μm. E CD276 expressed in SK-MES-1, H520, H1703, and B2B shown by qRT-PCR. F B7H3 localization in H1703/H520/SK-MES-1 shown by IF (×40 magnification). Representative images are shown. Scale bars = 100 μm. G B7H3 expression on the cell membrane of H1703 and H520 shown by flow cytometry. * P < 0.05; ** P < 0.01; *** P < 0.001. ns, not significant. Variables are presented as mean ± SD.

Article Snippet: LUSC cell lines (H1703, H520, and SK-MES-1) and the pulmonary epithelial cell BEAS-2B (B2B) were purchased from China Center Type Culture Collection (CCTCC, Shanghai) and cultured in RPMI-1640 (GIBCO, USA) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA) at 37°C and 5% CO2.

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Membrane, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma

doi: 10.1038/s41419-023-06376-w

Figure Lengend Snippet: A Normalized cell index of B7H3-DAP12-CAR-T co-cultured with target cells at different ratios (E:T = 0:1, 1:1, 2:1, 5:1) in 25 or 100 μM Met within 28 h. B Normalized cell index of LUSC cell lines at 10, 25, 50, 75, or 100 μM Met within 24 h. C Normalized cell index of B7H3-DAP12-CAR-T co-cultured with target cells (E:T = 0:1 or 2:1) at 25, 50, 75, or 100 μM Met. D IL-2 and IFN-γ secreted by B7H3-DAP12-CAR-T co-cultured with target tumor cells (2 × 10 5 ) at E:T = 2:1 for 48 h in 25, 50, 75, or 100 μM Met measured by ELISA. E Expression of cytokine-associated genes in CAR-T co-cultured with 1703-B7H3 (E:T = 1:1) at 25 or 100 Met shown by qRT-PCR. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Variables are presented as mean ± SD.

Article Snippet: LUSC cell lines (H1703, H520, and SK-MES-1) and the pulmonary epithelial cell BEAS-2B (B2B) were purchased from China Center Type Culture Collection (CCTCC, Shanghai) and cultured in RPMI-1640 (GIBCO, USA) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA) at 37°C and 5% CO2.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma

doi: 10.1038/s41419-023-06376-w

Figure Lengend Snippet: A Expression of classical Met transporters (SLC7A5, SLC7A6, SLC7A7, SLC7A8,SLC38A1, SLC38A2, SLC43A2) in LUSC ( n = 502) relative to normal adjacent tissues ( n = 51) based on TCGA shown by the heatmap and bar chart. B Expression of SLC7A5 among 26 types of LUSC cell lines based on DepMap. C A Venn diagram consisting of SLCs highly expressed in LUSC based on TCGA, SLCs highly expressed in LUSC cell lines based on DepMap, and SLCs negatively associated with the overall survival of LUSC patients based on TCGA. D The relationship between SLC7A5 and CD8 + T cells (R = –0.224, P < 0.001) in LUSC analyzed by Timer2.0. E SLC7A5 expressed in SK-MES-1, H520, H1703, and B2B shown by qRT-PCR. F SLC7A5 expressed in SK-MES-1, H520, H1703, and B2B shown by WB (left); SLC7A5 expression in 1703-B7H3 and CAR-T after co-cultured (E:T = 2:1) for 48 h in 25 or 100 μM shown by WB (right). G After culturing for 2 days, Met concentration in supernatant of 1 × 10 5 LUSC cells with SLC7A5 up- or down-regulated detected by ELISA. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Variables are presented as mean ± SD.

Article Snippet: LUSC cell lines (H1703, H520, and SK-MES-1) and the pulmonary epithelial cell BEAS-2B (B2B) were purchased from China Center Type Culture Collection (CCTCC, Shanghai) and cultured in RPMI-1640 (GIBCO, USA) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA) at 37°C and 5% CO2.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: SOX9 regulates expression of Foxc1 . (A) SOX9 expression was induced in mouse embryonic stem cells containing a doxycycline (Dox)-inducible cassette. Expression was induced for 48h by removal of Dox. Expression of Foxc1, Foxc2 Col2a and Sox9 mRNA was determined by qRT-PCR. Data are presented from three independent experiments. Error bars represent standard deviation. (B) SOX9 binding to regulatory elements in Col2a and Foxc1 was determined by Chromatin Immunoprecipitation (ChIP) in ATDC5 chondrocyte cells. Four Sox9 binding sites in the regulatory region of Foxc1 (Distal A-D) were identified from previous ChIP-seq experiments (Ohba et al 2015). (C) Foxc1 Distal regulatory elements were cloned into pGL4-luciferase reporters and transfected along with Sox9 in ATDC5 cells. Only Foxc1-Distal C was activated by Sox9 . * p-value <0.05.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Clone Assay, Luciferase, Transfection

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Elevated Foxc1 promotes chondrocyte differentiation of mouse embryonic stem cells (mES). Schematic of chondrocyte differentiation protocol for mES cells (Kawaguchi et al 2005). (B) Expression of FOXC1 was confirmed in mES cells containing a Dox-inducible flag-tagged Foxc1 expression cassette. (C) Chondrocyte differentiation in mES cells was monitored by measuring expression of Sox9, Sox6, Col2a, Runx2 and ColX expression. Data presented are the means from three biological replicates. Error bars represent standard deviation. * p-value <0.05

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Loss of Foxc1 expression in ATDC5 cells alters chondrocyte differentiation. (A) Deletion of Foxc1 expression was achieved through Crispr mutagenesis. Reduced protein levels observed in Foxc1 mutant cells (crFOXC1). (B) Wild type and crFOXC1 cells were differentiated for 21 days and chondrogenesis measured by Alcian Blue staining. The number of chondrogenic nodules were counted in wild type and mutant cells. (C) Levels of chondrocyte-expressed genes are affected in Foxc1 mutant ATDC5 cells after 21 days of differentiation.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, CRISPR, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Foxc1 and Foxc2 are expressed in the perichondrium and resting zone of the growth plate. (A) Localization of Foxc1 and Foxc2 expression was determined in the 14.5 dpc hind limb by in situ hybridization. (B)Foxc1 and (C) Foxc2 mRNA expression in the proximal tibia at 16.5 dpc was abundant in the perichondrium. (D). Foxc1 and Foxc2 mRNA displayed both overlapping and distinct expression patterns in the developing limb. (E). Foxc1 transcripts (Red) were detected in the perichondrium and resting zone, while Foxc2(Green) was mainly expressed in the perichondrium. Low levels of Foxc1 and Foxc2 expression were detected in the proliferative zone (F) and hypertrophic zone (G).

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Col2-cre ablation of Foxc1 and Foxc2 expression in the developing limb. (A) (B) ROSA26 tm4(ACTB-tdTomato,-EGFP) (mTmG) mice were crossed to Col2-Cre mice to monitor Cre activity in the developing limb at 14.5 dpc. EGFP was only detected in limb of mice containing the Col2-cre transgene. (C)(D) To create chondrocyte-specific Foxc1 and Foxc2 mutant mice, Col2-cre mice were crossed with homozygous “floxed” Foxc1 and Foxc2 mice. No expression of Foxc1 and Foxc2 mRNA was detected by in situ hybridization in the developing humerus or proximal tibia in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mice at 16.5 dpc.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Activity Assay, Mutagenesis, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Col2-cre deletion of Foxc1 and Foxc2 causes skeletal hypoplasia. (A). Alizarin red and Alcian blue skeleton preps from 18.5 dpc embryos. Control embryos lack the Col2-cre transgene. (B) Disrupted endochondral ossification can be observed in the axial and appendicular skeleton of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos. (C)Length of ossification is reduced in the tibia at 18.5 dpc of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mice is reduced.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Disrupted growth plate organization in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants. (A) Hematoxylin-Eosin staining of the 16.5 dpc proximal tibia growth plate. Enlarged sections are depicted with coloured boxes. (B) Length of the resting, proliferative and hypertrophic zone were measured in control (CON) or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ (C1 ΔΔ ;C2 ΔΔ ) growth plate. (C) Chondrocyte proliferation in control of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ was measured by Ki67 immunofluorescence.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Staining, Control, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Loss of Foxc1 and Foxc2 function in chondrocytes results in a general reduction of mRNA levels of genes expressed throughout endochondral ossification. (A) RNA from the rib cage from control or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos at 16.5 dpc was isolated and gene expression monitored by qRT-PCR. (B) Heat map of the top 25 genes down regulated in 16.5 dpc Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ rib RNA as determined by RNA-seq analysis. (C) Functional annotation genes down regulated Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control, Isolation, Gene Expression, Quantitative RT-PCR, RNA Sequencing, Functional Assay

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Altered chondrocyte differentiation in Col2-cre;Foxc1D/D;Foxc2D/D embryos. Gene expression patterns markers of chondrocyte differentiation were monitored in the proximal tibia growth plate at 16.5 dpc in control or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos. (A) SOX9 immunofluorescence (IF) SOX6 IF (C) Fgfr3 in situ hybridization (D) Ihh ISH (E) RUNX2 IF (F) COLX IF; (G) MMP13 IF. Length of RUNX2 (H) and COLX (I) positive cells in control vs Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ growth plates. Scale bar 200 mm.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Gene Expression, Control, Immunofluorescence, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Impaired mineralization in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants. (A,A’-B,B’) Mineralization in the primary ossification center of control and Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos determined by Von Kossa staining. (C-D) Levels of OSTEOPONTIN (Red) and OSTERIX protein (E,F,) COLLAGEN I and MMP13 localization (G-H) VEGFA localization.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control, Staining