abc vectorstain procedure  (Vector Laboratories)

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: Expression and phosphorylation of Stat3 and Stat5. (A–C) Stat3 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 −/− mice. (A) pStat3 was detected in sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) Btn1a1 −/− (lactation day 10, L10) and (d) Btn1a1 +/+ (involution day 1, Inv1) mice by the ABC VectorStain procedure as described in Materials and Methods. Representative examples of each genotype (3–6 sections each from three mice/genotype). Bar 100 µm. (B) Expression of Stat3 and phosphorylated Stat3 monitored by immunoblot of THs (60 µg protein/lane; 10% polyacrylamide) from 5 Btn1a1 +/+ , 7 Btn1a1 −/− mice at lactation days 9‐11 (L9‐11) and three each, Btn1a1 ++ mice at involution days 1 and 2 (Inv1 and Inv 2, respectively). pStat3 was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y705 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat3 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐ C‐terminal Stat3‐peptide antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (C) Triplicate blots as in B were quantified and data normalized to the amount of pStat3 in the Inv1 samples (set to 100%). pStat3/ Stat3 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA). (D–F) Stat5 is phosphorylated and accumulates in the nucleus at peak lactation (day 10) in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice. (D) pStat5 was detected in sections of paraffin‐embedded mammary gland as in A–C. Representative examples of each genotype (3–5 sections each from three mice/genotype). Alveoli denuded of epithelial cells (alveolar “ghosts”) discussed in the text are marked by asterisks in the knockout sample. Bar 100 µm. (E) Expression of Stat5 and phosphorylated Stat5 monitored by immunoblot of THs as in B above. Phosphorylated Stat5 (pStat5) was detected with a 1000‐fold dilution of rabbit anti‐peptide‐Y694 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. Total Stat5 was detected on the same stripped blot with a 1000‐fold dilution of rabbit anti‐peptide Stat5 antibody, followed by a 1000‐fold dilution of goat anti‐rabbit IgG‐HRP. (F) Duplicate blots as in E were quantified and data normalized to the amount of pStat5 in the Btn1a1 ++ L10 samples (set to 100%). pStat5/ Stat5 ratios are given below the graph. Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Western Blot, Knock-Out

Journal: FASEB BioAdvances

Article Title: The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

doi: 10.1096/fba.2021-00059

Figure Lengend Snippet: The number (%) of mitotic epithelial cells increases at peak lactation in Btn1a1 −/− mice. (A) Ki67 was detected in sections of paraffin‐embedded mammary gland from (a) Btn1a1 +/+ , (b) Btn1a1 +/− , and (c) Btn1a1 −/− (lactation day 10, L10) by the ABC VectorStain procedure, as described in Materials and Methods. The number of Ki67‐positive nuclei was significantly increased in luminal Btn1a1 −/− cells compared with cells in Btn1a1 +/+ and Btn1a1 +/− mice (arrowheads). Representative examples of each genotype (4–6 sections, each from five mice/genotype). Bar 100 µm. (B) Section of embryonic day‐18 mouse mammary gland used as a positive control for Ki67 staining (1 of 5 examples). (C) Comparison of the number (%) of Ki67‐positive luminal cells at day 10 of lactation in Btn1a1 +/+ , Btn1a1 +/− , and Btn1a1 −/− mice (respective number of cells counted/genotype, 2,979, 2,529, and 1,418). Columns with different letters are statistically different from each other. Means ± SEM, p < 0.05, F‐test (ANOVA)

Article Snippet: Sections (7 µm) were dewaxed with Histoclear (HS‐200, National Diagnostics), rehydrated through a descending series of ethanol/water mixtures, and then immersed in 10 mM citrate buffer, pH 6.0 to retrieve antigen by heating in a pressure cooker for 1 min. Endogenous peroxidase was quenched by incubating the sections in 0.6% H 2 O 2 / 100% methanol for 30 min and the sections then rehydrated in water for 5 min. After blocking with 10% normal goat serum for 1 h, sections were incubated with 1‐ to 100‐fold dilutions of primary antibodies to either pStat3, pStat5, or Ki67 overnight at 4°C, followed by 1‐ to 50‐fold dilutions of mouse biotinylated anti‐rabbit IgG and avidin‐HRP, according to the ABC VectorStain procedure (Vector Laboratories, Burlingame, CA).

Techniques: Positive Control, Staining