immunohistochemical analysis post heat mediated antigen retrieval  (Vector Laboratories)

Journal: Journal of cell science

Article Title: FMRP protects the lung from xenobiotic stress by facilitating the integrated stress response.

doi: 10.1242/jcs.258652

Figure Lengend Snippet: Fig. 1. FMRP is expressed in the airways and more broadly, and protects airway club cells from Nap-induced stress. (A–D) FMRP expression in the murine lung. (A) Tiled image showing FMRP immunostaining (green, white arrows) in the airway epithelium (demarcated by white dashed lines) and in the parenchyma of the murine lung. The airways are identified by expression of the club cell (CC) marker Scgb1a1 (white, inset) and of the ciliated cell marker acetylated tubulin (Ac-tub, red, inset). (B) Tiled image showing FMRP immunostaining in Fmr1 knockout (Fmr1 KO) mice. Note the absence of FMRP (green) in both airways (demarcated by white dashed lines, inset) and parenchyma. (C,D) High-resolution image of FMRP immunostaining (green) in airway epithelial cells. Here, CCs are shown in white (white arrow in inset) and ciliated cells are in red (white arrowhead in inset) in wild type (C) and Fmr1 KO (D). (E–H) Susceptibility of CCs to Nap injury in control and Fmr1 KO. (E) Schematic showing regimen for Nap injury. (F) Frequencies of Scgb1a1+ cells in wild type (black circles) and Fmr1 KO (gray squares) from uninjured (Un) and Nap-injured mice at different timepoints post injury. Each data point in the scatter plot represents multiple sections from a single animal (n=3 mice) with mean±s.e.m. (G,H) Expression of markers of oxidative (4HNE) and genotoxic (γ-H2AX) stress in airways from wild-type and Fmr1 KO mice prior to and post Nap injury. Note white arrowheads showing ciliated cells and white arrows showing CCs on the airways and in insets (counterstained with Scgb1a1 antisera, white). (G i–vi) 4HNE immunostaining (green) in the airways of wild-type (i,iii,v) and Fmr1 KO (ii,iv,vi) mice prior to and post Nap injury. (H i–vi) γ-H2AX immunostaining (red) in the airways of control (i,iii,v) and Fmr1 KO (ii,iv,vi) mice prior to and post Nap injury. Asterisks show cilia of ciliated cells marked with γ-H2AX (H i–vi). See also Fig. S1. Statistical significance was assessed by an unpaired two-tailed t-test: *P<0.05; **P<0.01; ***P<0.001. The changes in the two groups over time, across genotype and interaction parameters were also assessed by two-way ANOVA and found to be statistically significant. For Shapiro–Wilk normality test and two-way ANOVA, see Table S1. Scale bars: 20 μm.

Article Snippet: Fixed lungs were subsequently embedded in paraffin, sectioned (5 μm) and processed for immunohistochemical analysis post heat-mediated antigen retrieval at pH 6.0 (Vector Labs, USA, H-3300) except for sections stained with anti-SOD1 antisera, which were subject to antigen retrieval at pH 9.0 (Vector Labs, USA, H-3301).

Techniques: Expressing, Immunostaining, Marker, Knock-Out, Control, Two Tailed Test

Journal: Journal of cell science

Article Title: FMRP protects the lung from xenobiotic stress by facilitating the integrated stress response.

doi: 10.1242/jcs.258652

Figure Lengend Snippet: Fig. 2. FMRP-deficient club-cell-like C22 cells are susceptible to Nap-induced stress. (A–D) Phenotypic characterization of C22 cells. (A) Scgb1a1 immunostaining (white) in C22 cells. Inset shows Scgb1a1 staining alone in C22 cells from the same field. (B) Cyp2f2 (orange) immunostaining in C22 cells. Inset shows Cyp2f2 staining alone in C22 cells from the same field. See also Fig. S2. (C,D) FMRP immunostaining (green) in C22 cells treated with scrambled siRNA (C) and in C22 cells treated with Fmr1 siRNA (D). (E–J) Susceptibility of C22 cells to Nap [control is scrambled siRNA-treated (Sc), and Fmr1 siRNA-treated (Si)]. (E) Schematic showing regimen for Nap injury. (F–I) Expression of markers of oxidative (4HNE) and genotoxic (γ-H2AX) stress in Sc and Si cells prior to and post Nap. (Fi–vi) 4HNE immunostaining (green) in Sc and Si cells prior to and post Nap. (G) Quantification of 4HNE immunofluorescence per cell in Sc and Si cells prior to and post Nap. Un, uninjured. (Hi–vi) γ-H2AX immunostaining (red) in Sc and Si cells prior to and post Nap. (I) Quantification of γ-H2AX immunofluorescence per cell in Sc and Si cells post Nap. (J) Cytotoxicity of Nap in Sc and Si cells 24 h post Nap (n=3 experiments). For immunofluorescence analysis, ≥25 cells were analyzed per timepoint per experiment, n=3 experiments. Graphical data represent mean±s.e.m. Black circles, Sc; gray squares, Si. *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed t-test). For normality test and two-way ANOVA, see Table S2. Scale bars: 5 μm.

Article Snippet: Fixed lungs were subsequently embedded in paraffin, sectioned (5 μm) and processed for immunohistochemical analysis post heat-mediated antigen retrieval at pH 6.0 (Vector Labs, USA, H-3300) except for sections stained with anti-SOD1 antisera, which were subject to antigen retrieval at pH 9.0 (Vector Labs, USA, H-3301).

Techniques: Immunostaining, Staining, Control, Expressing, Immunofluorescence, Two Tailed Test

Journal: Journal of cell science

Article Title: FMRP protects the lung from xenobiotic stress by facilitating the integrated stress response.

doi: 10.1242/jcs.258652

Figure Lengend Snippet: Fig. 3. FMRP-deficient C22 cells fail to upregulate the integrated stress response and to induce ATF4, essential for protection from Nap-induced stress. (A) Western blot-based quantification of phospho-eIF2α:eIF2α ratios in Sc and Si cells prior to and post Nap treatment (n=5 experiments). See Fig. S3 for representative blots used for quantification. Un, uninjured. (Bi–viii) ATF4 immunostaining (white) in Sc (i,iii,v,vii) and Si (ii,iv,vi,viii) cells prior to and post Nap. Note nuclear accumulation of ATF4 in Sc cells by 6 h post Nap (inset). (C) Quantification of ATF4 immunofluorescence per cell in Sc and Si cells prior to and post Nap (n=5 experiments). (D–G) Susceptibility of C22 cells to Nap in control (scrambled siRNA-treated, Sc) and Atf4 siRNA-treated (Si) cells. (Di–xii) Analysis of ATF4 levels (white) and FMRP levels (green) in Sc and Si cells prior to and post Nap treatment. Immunostaining for ATF4 (white) and FMRP (green) in Sc (i,ii,v,vi,ix,x) and Si (iii,iv,vii,viii,xi,xii) cells. (E) Quantification of 4HNE immunofluorescence per cell in Sc and Si cells prior to and post Nap. See Fig. S3 for representative images. (F) Quantification of γ-H2AX immunofluorescence per cell in Sc and Si. See Fig. S3 for representative images. (G) Cytotoxicity of Nap in Sc and Si cells 24 h post Nap exposure (n=3 experiments). For immunofluorescence analysis, ≥25 cells were analyzed per timepoint per experiment. Graphical data represent mean±s.e.m. Black circles, Sc; gray squares, Si. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (unpaired two-tailed t-test). For normality test and two-way ANOVA, see Table S3. Scale bars: 5 μm.

Article Snippet: Fixed lungs were subsequently embedded in paraffin, sectioned (5 μm) and processed for immunohistochemical analysis post heat-mediated antigen retrieval at pH 6.0 (Vector Labs, USA, H-3300) except for sections stained with anti-SOD1 antisera, which were subject to antigen retrieval at pH 9.0 (Vector Labs, USA, H-3301).

Techniques: Western Blot, Immunostaining, Immunofluorescence, Control, Two Tailed Test

Journal: Journal of cell science

Article Title: FMRP protects the lung from xenobiotic stress by facilitating the integrated stress response.

doi: 10.1242/jcs.258652

Figure Lengend Snippet: Fig. 4. FMRP is expressed in the human airways and protects human bronchial BEAS-2B cells from PQ-induced stress. (A–C) FMRP expression in the human lung and in BEAS-2B cells, a cell line derived from the human bronchial epithelium. (Ai–iv) FMRP immunostaining (green) in the distal airways of the human lung. (Ai,ii,iv) Stained section showing FMRP expression in airway non-ciliated cells [Scgb1a1+ (white), white arrows; Scgb1a1−, yellow arrows] and ciliated cells (red, red arrow). The white dashed line indicates airway epithelium. The boxed area in i is shown at higher magnification in ii and iv. Negative control [secondary antibody (Sec) alone] for FMRP immunostaining is shown in iii. (B) FMRP immunostaining (green) of BEAS-2B cells (control, scrambled siRNA- treated, Sc). (C) FMRP immunostaining (green) of FMR1 siRNA-treated BEAS-2B cells. (D–I) Susceptibility of BEAS-2B cells to PQ injury in control (scrambled siRNA-treated, Sc) and FMR1 siRNA-treated (Si) cells. (D) Schematic showing regimen for PQ injury. (E–H) Expression of markers of oxidative (4HNE) and genotoxic (γ-H2AX) stress in Sc and Si cells prior to and post PQ. Un, uninjured. (Ei–x) 4HNE immunostaining (green) in Sc and Si cells prior to and post PQ. (F) Quantification of 4HNE immunofluorescence per cell in Sc and Si cells prior to and post PQ (n=3 experiments). (Gi–x) γ-H2AX immunostaining (red) in Sc and Si cells prior to and post PQ. (H) Quantification of γ-H2AX immunofluorescence per cell in Sc and Si cells prior to and post PQ. (I) Cytotoxicity of PQ in Sc and Si cells 24 h post PQ exposure (n=3 experiments). For immunofluorescence analysis, ≥25 cells were analyzed per timepoint per experiment. Graphical data represent mean±s.e.m. Black circles, Sc; gray squares, Si. *P<0.05; **P<0.01; ***P<0.001 (unpaired two-tailed t-test). For normality tests and two-way ANOVA, see Table S4. Scale bars: 5 μm.

Article Snippet: Fixed lungs were subsequently embedded in paraffin, sectioned (5 μm) and processed for immunohistochemical analysis post heat-mediated antigen retrieval at pH 6.0 (Vector Labs, USA, H-3300) except for sections stained with anti-SOD1 antisera, which were subject to antigen retrieval at pH 9.0 (Vector Labs, USA, H-3301).

Techniques: Expressing, Derivative Assay, Immunostaining, Staining, Negative Control, Control, Immunofluorescence, Two Tailed Test

Journal: Journal of cell science

Article Title: FMRP protects the lung from xenobiotic stress by facilitating the integrated stress response.

doi: 10.1242/jcs.258652

Figure Lengend Snippet: Fig. 5. FMRP-deficient BEAS-2B cells fail to upregulate the integrated stress response and to induce ATF4, essential for protection from PQ-induced stress. (A) Western blot-based quantification of phospho-eIF2α:eIF2α ratios in Sc and Si cells prior to and post PQ treatment (n=5 experiments). Un, uninjured. See Fig. S4A,B for representative blots used for quantification. (B,C) Analysis of ATF4 prior to and post PQ. (Bi–viii) ATF4 immunostaining (white) in Sc (i,iii,v,vii) and Si (ii,iv,vi,viii) cells prior to and post PQ treatment. Note nuclear accumulation of ATF4 in Sc cells by 6 h post PQ treatment (inset). (C) Quantification of ATF4 immunofluorescence per cell in Sc and Si cells prior to and post PQ (n=5 experiments). (D–G) Susceptibility of BEAS-2B cells to PQ in control (scrambled siRNA- treated, Sc) and ATF4 siRNA-treated (Si) cells. (Di–xii) Analysis of ATF4 levels (white) and FMRP levels (green) in Sc (i,ii,v,vi,ix,x) and Si (iii,iv,vii,viii,xi,xii) cells prior to and post PQ treatment. (E) Quantification of 4HNE immunofluorescence per cell in Sc and Si. See Fig. S4G for representative images. (F) Quantification of γ-H2AX immunofluorescence per cell in Sc and Si. See Fig. S4H for representative images. (G) Cytotoxicity of PQ in Sc and Si cells 24 h post PQ treatment (n=3 experiments). For immunofluorescence analysis, ≥25 cells were analyzed per timepoint per experiment. Graphical data represent mean±s.e.m. Black circles, Sc; gray squares, Si. **P<0.01; ***P<0.001; ****P<0.0001 (unpaired two-tailed t-test). For normality test and two-way ANOVA, see Table S5. Scale bars: 5 μm.

Article Snippet: Fixed lungs were subsequently embedded in paraffin, sectioned (5 μm) and processed for immunohistochemical analysis post heat-mediated antigen retrieval at pH 6.0 (Vector Labs, USA, H-3300) except for sections stained with anti-SOD1 antisera, which were subject to antigen retrieval at pH 9.0 (Vector Labs, USA, H-3301).

Techniques: Western Blot, Immunostaining, Immunofluorescence, Control, Two Tailed Test