Biotinylated goat anti rabbit igg

Immunohistochemisty was performed on paraffin embedded liver tissues using antibodies specific to PP2Ac (Abcam, Cambridge, UK), α-SMA (Abcam, Cambridge, UK), and PCNA (Abcam, Cambridge, UK). Dewaxed and rehydrated paraffin-embedded sections were incubated with methanol: hydrogen peroxide (1 : 10) to block endogenous peroxidase activity and then were washed in Tris-buffered saline (pH 7.6). The slides were then incubated with the primary antibodies overnight at room temperature. After rinsing with Tris-buffered saline for 15 min, tissues were incubated with secondary antibody (biotinylated goat anti-rabbit IgG, Sigma). Sections were then washed and incubated with the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) for 45 min. Staining was developed using 3,3-diaminobenzidine (2.5 mg/mL) followed by counterstaining with Mayer's hematoxylin. Five high-power fields (400x magnification) were randomly selected within each slide. Data are expressed as the average percentage of positive staining cells.

Immunohistochemistry was performed on paraffin embedded colon tissues using specific antibodies (summarized in S1 Table). Dewaxed and rehydrated paraffin-embedded sections were incubated with methanol: hydrogen peroxide (1:10) to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6). The slides were then incubated with the primary antibodies overnight at room temperature. After rinsing with Tris-buffered saline for 15 min, tissues were incubated with secondary antibody (biotinylated goat anti-rabbit IgG, Sigma). Sections were then washed and incubated with the Vectastain Elite ABC reagent (Vector Laboratories. Burlingame, CA) for 45 min. Staining was developed using 3, 3-diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer's hematoxylin. 10 high-power fields (400× magnification) were randomly selected within each slide. Data are expressed as the average percentage of positive staining cells.

Immunohistochemical staining was performed as described previously52 (link) with Affinity-purified goat anti-mouse type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL, USA) and polyclonal Rabbit anti-Ki67 (Abcam, Cambridge, UK). Briefly, dewaxed and rehydrated paraffin-embedded sections were incubated with methanol:hydrogen peroxide (1:10) to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6). The slides were then incubated with the primary antibodies overnight at room temperature. After rinsing with Tris-buffered saline for 15 minutes, tissues were incubated with secondary antibody (biotinylated goat anti-rabbit IgG diluted 1:200, Sigma-Aldrich, St. Louis, MO). Sections were then washed and incubated with the Vectastain Elite ABC reagent (Vector Laboratories, Inc. Ontario, Canada) for 45 minutes. Staining was developed using 3,3-diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer’s Hematoxylin.

Immunohistochemical staining was performed for osterix, SOD1, SOD2, FOXO3a, β-galactosidase (β-gal), p16 and p21 using the avidin-biotin-peroxidase complex technique as described 12 . Briefly, dewaxed and rehydrated paraffin-embedded sections were incubated with 6% hydrogen peroxide to block endogenous peroxidase activity and then washed in PBS (pH 7.6). The slides were then incubated at 4°C overnight with the primary antibodies to osterix (Abcam), SOD1/2 (Abcam), FOXO3a (Abcam), β-gal (Abcam), p16 (Abcam), and p21 (Santa Cluz). After rinsing with PBS for 15 min, tissues were incubated with secondary antibody (biotinylated goat anti-rabbit IgG and goat anti-mouse IgG, Sigma). Sections were then washed and incubated with the Vectastain Elite ABC reagent (Vector Laboratories) for 30 min. Staining was done using 3,3-diaminobenzidine (2.5mg/ml) followed by counterstaining with Mayer's hematoxylin.

Immunocytochemistry for CC3 and β‐catenin was performed to analyse cell death after 24‐hr stimulation with Wnt ± H₂O₂ or to analyse β‐catenin localization after 30 min. VSMCs were fixed with 3% paraformaldehyde/PBS and permeabilized with 0.1%–0.2% Triton X‐100/PBS. After blocking with 20% goat serum/PBS, 1 µg/ml CC3 antibody (AF835; R&D Systems) in 1% BSA/PBS or 2.5 µg/ml β‐catenin antibody (610154; BD Transduction Laboratories, Oxford, UK) in PBS was added overnight at 4°C. Bound antibodies were detected with biotinylated goat anti‐rabbit IgG (B7389; Sigma‐Aldrich) or biotinylated goat anti‐mouse IgG (BA9200; Vector Laboratories, Peterborough, UK) and then DyLight‐488 Streptavidin (SA‐5488‐1; Vector Laboratories) diluted 1:200 in PBS. Coverslips were mounted in ProLong Gold and DAPI (P36931; Invitrogen, Paisley, UK).

Immunohistochemical staining for type I collagen (Col-I) was performed using an affinity-purified goat anti-mouse Col-I antibody (Southern Biotechnology Associates) as described previously⁽¹⁷⁾. Briefly, dewaxed and rehydrated paraffin-embedded sections were incubated with methanol–H₂O₂ (1:10) to block endogenous peroxidase activity, and then washed in Tris-buffered saline (pH 7·6). The slides were incubated with the primary antibody overnight at room temperature. After rinsing with Tris-buffered saline for 15 min, tissues were incubated with a secondary antibody (biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgG; Sigma). Next, the sections were washed and incubated with the VECTASTAIN Elite ABC reagent (Vector Laboratories) for 45 min. Staining was developed using 3,3′-diaminobenzidine (2·5 mg/ml) followed by counterstaining with Mayer's haematoxylin.

Lung lobes were collected and fixed with 4% paraformaldehyde overnight and embedded in paraffin. Hematoxylin and eosin (HE)-stained tissues were assessed via pathological analysis. Lung injury was estimated by the percentage of the lesion area in the total lung area using an ImagePro macro. Immunohistochemical staining of α-SMA, vimentin, E-cadherin, ZO-1 and integrin αvβ6 was performed using tissue sections that were dewaxed and rehydrated. Antigen retrieval was performed using proteinase K and hot citric acid buffer treatment as needed. The restored sections were incubated with primary antibodies overnight at 4°C. After rinsing with Tris-buffered saline for 15 min, sections were incubated with secondary antibody (biotinylated goat anti-rabbit IgG, Sigma). Sections were then washed and incubated with Vectastain Elite ABC reagent (Vector Laboratories, Burlington, ON, Canada) for 45 min. Staining was developed using 3,3-diaminobenzidine (2.5 mg/mL) followed by counterstaining with Mayer’s hematoxylin. Images were taken with a Leica Microsystems Ltd. microscope and subsequently analyzed independently with Image-Pro Plus 6 by two pathologists who had no experimental information.