Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, Schematic of alveolar organoid culture utilized for single-cell RNA-seq. b, Uniform manifold approximation and projection (UMAP) visualization of epithelial populations in cultured alveolar organoids (4,573 cells). AEC2 (green, 3,303 cells) – alveolar epithelial type-2 cells, AEC2-proliferating (purple, 696 cells) – proliferating alveolar epithelial type-2 cells, AEC1 (yellow, 262 cells) – alveolar epithelial type-1 cells, new cell states : Lgals3+ cells (blue, 184 cells) and Ctgf+ cells (red, 128 cells) c, UMAP plots show the expression of indicated genes in epithelial populations in cultured alveolar organoids. Dotted circles in b and c indicate the transitional cell states. d, Volcano plot shows specific genes enriched in Ctgf+ (n=128 cells) and Lgals3+ (n=184 cells) transitional cell states. Wilcoxon rank sum test was used for the statistical analysis. e, Schematic of alveolar organoid culture using fibroblasts and AEC2 cells. f, Immunostaining for PATS markers in alveolar organoids. Left panel - LGALS3 (green), SFTPC (red) and AGER (grey); middle panel - CLDN4 (green), Sftpc-tdt (red) and AGER (grey); right panel - SOX4 (green), SFTPC (red) and AGER (grey). Image is representative of 30 organoids from three mice. DAPI stains nuclei (blue). Scale bars indicate 20 μm.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: RNA Sequencing Assay, Cell Culture, Expressing, Immunostaining

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, Schematic of bleomycin-induced lung injury in Ctgf-GFP mice. b-c, Immunostaining for PATS markers. b, Ctgf-GFP (green), CLDN4 (red) and LGALS3 (grey), c, Ctgf-GFP (green), SFN (red) and LGALS3 (grey) in control lung (upper panel) and bleomycin-treated lungs collected on day 12 after injury (lower panel). Scale bars indicate 30 μm. d, Experimental design to ablate AEC1s in Ager-CreER;R26R-DTR mouse model. Mice were administered tamoxifen (Tmx) followed by diphtheria toxin (DT) and tissues collected on day 6. e, Immunostaining for CLDN4 (green) and LGALS3 (grey) in control (upper panel) and AEC1-ablated lungs (lower panel). Scale bars indicate 20 μm. f, Quantification of elongated LGALS3+ cells in control and AEC1-ablated lungs. ***p<0.0008 (two-tailed, un-paired student’s t-test). n=3 mice. g, Experimental workflow for sequential administration of tamoxifen followed by bleomycin injury and tissue collection for analysis using Sftpc-CreER;R26R-tdTomato mice. h-j, Immunostaining for PATS markers in Sftpc-lineage labeled cell in control (upper panel) and bleomycin injured lungs (lower panel). h, SFTPC (green), Sftpc-tdt (red) and LGALS3 (grey), i, SFN (green), Sftpc-tdt (red) and AGER (grey), and j, CLDN4 (green), Sftpc-tdt (red) and AGER (grey). DAPI stains nuclei (blue). Scale bars indicate 30 μm. White box in merged image indicates region of single channel images. k, Time course of immunostaining for CLDN4 (green), Sftpc-tdt (red) and LGALS3 after bleomycin injury. Scale bars indicate 10 μm. l, Quantification of CLDN4+ cells (green), CLDN4+/LGALS3+ cells (blue) and LGALS3+ cells (red) at different times after injury. n=3 mice. m, Quantification of cell length of CLDN4+ cells (green), CLDN4+/LGALS3+ cells (blue) and LGALS3+ cells (red). *p =0.049 for green vs blue; *p =0.0235 for green vs red (two-tailed un-paired student’s t-test). n=30 cells from three mice. n, Schematic showing transition from AEC2 to AEC1 through different PATS subtypes. Data are from three independent experiments. In f, l, m, data are presented as mean ± s.e.m. Images from b, c, e, h, i, j, k are representative from three mice. h-k experiment was repeated three times independently with similar results.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Immunostaining, Two Tailed Test, Labeling

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a. Experimental workflow for sequential administration of tamoxifen followed by PBS or bleomycin (d0) administration and Nutlin-3a or DMSO treatment (d8–18) and tissue collection (d20) for analysis using Sftpc-CreER;R26R-tdTomato mice. b, Immunostaining for SFTPC (green), Sftpc-tdt (red) and AGER (grey) in PBS+Nutlin-3a (left panel), bleomycin+DMSO (middle panel) and bleomycin+Nutlin-3a (right panel) treated mice. Scale bar: 100 μm. c, Quantification of AGER+Sftpc-tdt+ cells in total Sftpc-tdt+ Cells. *p = 0.0201; (two-tailed, unpaired t-test). n=3 mice. d. Experimental workflow for sequential administration of tamoxifen to delete TP53 in AEC2 cells followed by bleomycin injury (d0) in Sftpc-CreER;R26R-tdTomato;Trp53fl/fl or control mice (Sftpc-CreER;R26R-tdTomato;Trp53+/+). e, Immunostaining for SFTPC (green), Sftpc-tdt (red) and AGER (grey) (upper panel) and CLDN4 (green), Sftpc-tdt (red) and LGALS3 (grey) (lower panel) in control (left panel) and Trp53 knockout (right panel) mice. Scale bar: 30 μm. f, Quantification of AGER+Sftpc-tdt+ cells in total Sftpc-tdt+. ****p<0.0001 (two-tailed, un-paired student’s t-test). n=3 mice. g, Quantification of CLDN4+ cells (green), CLDN4+/LGALS3+ cells (blue) and LGALS3+ cells (red) in bleomycin-treated lungs. **p=0.048 (green bars) and **p=0.0013 (red bars) (two-tailed, un-paired student’s t-test). n=3 mice. h, Immunostaining for CDKN1A (p21) (green), Sftpc-tdt (red) and SFN (grey) in control (upper panel) and Trp53 knockout mice treated with bleomycin (lower panel). White arrowheads indicate p21+Sftpc-tdt+ cells. Scale bar: 30 μm. i, Quantification of p21+Sftpc-tdt+ cells in total Sftpc-tdt+ cells. ***p<0.0001 (two-tailed, un-paired student’s t-test). n=3 mice. j, Schematic of bleomycin-induced lung injury in Sftpc-CreER;R26R-tdTomato;Ctgf-GFP mice followed by PATS sorting and ChIP analysis. k, ChIP enrichment for TRP53 (orange), H3K4me3 (active promoter; green) and H3K27ac (active enhancer; red) shown in IGV tracks in Ctgf, (left) Cdkn1a (middle) and Krt19 (right). Blue-shaded regions indicate promoter/enhancer. DAPI stains nuclei (blue). Insets on the right side in all panels show individual fluorescence channels of region indicated by dotted line boxes. Data are from three independent experiments and are presented as mean ± s.e.m. Images from b, e, h are representative from three mice repeated independently with similar results.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Immunostaining, Two Tailed Test, Knock-Out, Fluorescence

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, Experimental design to ablate AEC1 cells using Ager-CreER;R26R-DTR mouse model. b-e, Immunostaining for b) SFN (green) and LGALS3 (grey) or c) SFTPC (green) and LGALS3 (grey) or d) SFN (green) and KRT19 (red) or e) LGALS3 (green) and Ki67 (red) in control (left panel) and AEC1-ablated lungs on day 6 (middle panel) and day 28 (right panel). f, Schematic of AEC2 lineage tracing using Sftpc-CreER;R26R-tdTomato mice follow by pneumonectomy (PNX) and tissue collection on day 9. g, Immunostaining for SFN (green), Sftpc-tdt (red) and LGALS3 (grey) in center (left panel) and edge (right panel) of the lungs after pneumonectomy. h, immunostaining for CLDN4 (green), Sftpc-tdt (red) and LGALS3 (grey) (right panel) in center (left panel) and edge (right panel) of the lungs after pneumonectomy. DAPI stains nuclei (blue). Scale bars indicate 20 μm. Images in b-e, g and h are representative from three mice repeated independently with similar results.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Expressing, Immunostaining

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, UMAP of epithelial populations in cultured alveolar organoids (4,573 cells). Arrow indicates selected cell populations in the oval-shaped circle are shown in panel b. b, UMAP plots show the expression of indicated genes in the selected populations (oval-shaped circle in panel a, 574 cells). c, RNA velocity analysis for PATS and AEC1. Arrows indicate predicted lineage trajectories. d, Schematic representation of experimental design to sequentially administer bleomycin (injury) or PBS (control) followed by tamoxifen (to label Krt19-expressing cells) in Krt19-CreER;R26R-tdTomato mice. e. Immunostaining for SFTPC (green), Krt19-tdt (red) and AGER (grey). White arrows indicate AGER+Krt19-tdt+ cells. (Scale bar: 30 μm). f, Co-staining for SFN (green), Krt19-tdt (red) and AGER (grey). White arrows indicate SFN+Krt19-tdt+ cells. (Scale bar: 50 μm). g. Immunostaining for CLDN4 (green), Krt19-tdt (red) and LGALS3 (grey). White arrows indicate CLDN4+Krt19-tdt+ cells. Yellow arrowhead indicates LGALS3+Krt19-tdt+ cell. (Scale bar: 30 μm). e-g Control lungs are shown in left panels and bleomycin day 12 injured lungs are shown in right panels. DAPI stains nuclei (blue). White box in merged image indicates region of single channel images shown in left side. Images from e-g are representative from three mice repeated independently with similar results.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Cell Culture, Expressing, Immunostaining, Staining

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, Schematic of bleomycin-induced lung injury in Sftpc-CreER;R26RtdTomato mice. b, β-galactosidase staining in bleomycin injured and control lungs. c, Immunostaining for CDKN1A (p21) (green), Sftpc-tdt (red) and AGER (grey). d, Immunostaining for γH2AX (green), Sftpc-tdt (red) and AGER (grey) in control and bleomycin injured lungs. Yellow arrowheads indicate γH2AX+ cells. White dotted lines indicate AEC1 cells and red dotted lines outline tdt+ cells. e, Immunostaining for γH2AX (green), Sftpc-tdt (red) and LGALS3 (grey) in control (upper) and bleomycin-treated lung (lower). f, Quantification of LGALS3+ γH2AX+Sftpc-tdt+ in total γH2AX+Sftpc-tdt+ cells in control and bleomycin injured mice on day-10. Asterisks indicate *p<0.0318 (one-tailed, Mann-Whitney). n=3 mice. g, Schematic of AEC1 ablation using diphtheria toxin (DT). h, β-galactosidase staining in DT-treated and control lungs. i, Immunostaining for CDKN1A (p21) (green), LGALS3 (red) and AGER (grey). j, Immunostaining for γH2AX (green), LGALS3 (red) and AGER (white) in control (upper panel) and DT-treated lungs collected on day-6 after injury (lower panel). k, Quantification of LGALS3+γH2AX+ cells in total γH2AX+ cells in control and DT-treated lung. *p=0.0318. (one-tailed, Mann-Whitney). n=3 mice. l, Schematic of 2D culture of AEC2. m, Immunostaining for SFTPC-GFP (green), γH2AX (red) and AGER (grey) in 2D culture of AEC2. White arrows indicate cells with DNA damage marker. Insets on the right side in all panels show individual fluorescence channels of region indicated by dotted line boxes. DAPI stains nuclei (blue). Data are from three independent experiments and are presented as mean ± s.e.m. Images from b, c, d, e, h, i, j, m are representative from three mice repeated independently with similar results. Scale bar: 100μm in b and h; 50μm in c and d; 30μm in e and m; 20μm in i and j.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Staining, Immunostaining, One-tailed Test, MANN-WHITNEY, Marker, Fluorescence

Journal: Nature cell biology

Article Title: Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis

doi: 10.1038/s41556-020-0542-8

Figure Lengend Snippet: a, Heatmap shows expression of known target genes of indicated signalling pathways in AEC2, proliferating AEC2 (AEC2pro), PATS and AEC1 in alveolar organoids (4,573 cells) and LPS-treated murine lung (13,204 cells). Scale indicates z-score where red is high, and blue is low. b, Immunostaining for YAP (red), Ctgf-GFP (green) and LGALS3 (grey) in bleomycin-treated mouse lung (left) and AEC1-ablated lung (right). Arrowheads indicate YAP expression in Ctgf-GFP+ cells. DAPI stains nuclei (blue). Scale bars indicate 25μm. n=3 mice.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181–200, 1:500), CLDN4 (Invitrogen, 36–4800, 1:200; Proteintech, 16165–1-AR, 1:500), GFP (Novus Biologicals, NB100–1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5–31424, 1:250), SFN (Invitrogen, PA5–95056, 1:250, Proteintech, 66251–1-Ig, 1:500, Abcam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), gamma-H2AX (R&D, 4418-APC, 1:500 and Novus Biologicals, NB100–74435, 1:250), CDKN1A (p21) (Sigma, ZRB1141, 1:200, BD Bioscience, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1000), YAP (Cell Signaling Technology, 4912S, 1:250). β-galactosidase (X-gal) staining and Hematoxylin & Eosin (H&E) staining PFA-fixed frozen sections were incubated with X-gal staining buffer containing 1mg/ml of X-gal (Thermo, R0941), 5mM K 3 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 , 2mM MgCl 2 , 0.01% sodium deocycholate and 0.02% NP-40 at 37ºC overnight.

Techniques: Expressing, Immunostaining