Horseradish peroxidase hrp conjugated goat anti rabbit igg

Lung tissue sections (5 μm thick) were deparaffinized and treated with 3% H₂O₂–CH₃OH for 15 minutes to block endogenous peroxidase. Samples were submerged in citrate buffer (pH 6.0) in a microwave oven for antigen retrieval, blocked with 5% BSA for 30 minutes at room temperature and then incubated overnight with antibody HMGB1 (1:1000). After washing with PBS, slices were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (ZSGB‐Bio) at 37°C for 30 minutes and then stained with DAB detection system kit (ZSGB‐Bio). HMGB1 expression and localization in the lung were detected under light microscopy and analysed by image‐pro plus 6.0 software.
For immunofluorescence, lung tissue sections were incubated overnight with antibodies HMGB1 (1:600), CD68 (1:100) or F4/80 (1:100). After washing with PBS, sections were incubated with alexa 594‐labelled goat anti‐rabbit secondary antibody and alexa 488‐labelled goat anti‐mouse/rat secondary antibody for 1 hour at 37°C, and then 4, 6‐diamidino‐2‐phenylind‐ole dihydrochloride (DAPI) (Beyotime) were added for cellular nuclear staining. Co‐localization of HMGB1 and CD68 or F4/80 was evaluated with confocal microscopy (TCS‐SP8; Leica Microsystems).

Lung tissues were cut into 5 μm sections, incubated in 0.3% H₂O₂-CH₃OH for 15 min for blocking endogenous peroxidase activity, then treated with citrate buffer (pH 6.0) using a microwave oven for 15 min to retrieve antigen, and followed by blocking with 5% bovine serum albumin for 30 min at room temperature (RT). Subsequently, the tissues were incubated overnight with antibodies against TRPV4 (1:100, Alomone labs, Jerusalem, Israel), human gasdermin D (GSDMD) (1:200, Abcam, Cambridge, United Kingdom), mouse GSDMD (1:1,500, Bioss Biotechnology Co., Ltd, Beijing, China), human gasdermin D N-terminal fragment (GSDMD-N) (1:400, Abcam), and mouse gasdermin D C-terminal fragment (GSDMD-C) (1:400, Abcam). The tissues were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZSGB-Bio, Beijing, China) at 37°C for 30 min. Finally, slides were visualized by staining with a 3,3′-Diaminobenzidine (DAB) detection system kit (ZSGB-Bio). Images were photographed by a microscope and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, MD, United States).

Lithium chloride, pilocarpine, diazepam, scopolamine, and MK-801 were purchased from Sigma. Propofol was purchased from AstraZeneca. Rabbit polyclonal antibodies against mouse and human GABA_A receptor a1 subunits were purchased from Alomone. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and 3,3-Diaminobenzidine (DAB) were purchased from ZSBIO. Protein BCA assay kit, Western Blot Stripping Buffer, skimmed milk powder, nitrocellulose membrane, and Super ECL Plus were purchased from Beijing Applygen Technologies. Wide-range prestained protein marker (molecular weight standard: 6–200 kd) was purchased from New England Biolabs. Other assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute.

The rat ileum segments and brain fixed in 4% paraformaldehyde were embedded in paraffin and sectioned following routine procedures [77 (link)]. Slides were blocked in 1× Tris-buffered saline containing 3% bovine serum albumin (BSA), 2% serum, and 0.02% Tween 20 at room temperature for 30 min. The sections were incubated with the primary antibody against P-gp (1:400 for rat ileum and 1:100 for rat brain, Abcam, Cambridge, UK) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZSGB-Bio, Beijing, China) at room temperature for 2 h. The sections were then visualized using a diaminobenzidine solution. Between each of the aforementioned steps, the sections were washed using 1× Tris-buffered saline tween (TBST) three times for 5 min each. Finally, images were captured using a Nanozoomer S210 microscopic-resolution scanner equipped with Digital Pathology View 2.0 software (Hamamatsu Photonics, Shizuoka, Japan). Java-based image-processing and analysis software (Image-Pro Plus; Version 6.0.0.260; National Institutes of Health, Bethesda, MD, USA) was used to analyze the proportion of P-gp-stained area in the whole ileal or cerebral cortex section of each rat in each group, respectively.

Lung tissue sections (5 μm thick) were deparaffinized, and treated with 3% H₂O₂-CH₃OH for 15 min to block endogenous peroxidase. Samples were submerged in citrate buffer (pH 6.0) in a microwave oven for antigen retrieval, blocked with 5% BSA for 30 min at room temperature and then incubated overnight with antibody F4/80 (1:1000), iNOS (1:100), CD163 (1:50), MMP9 (1:100), and MMP12 (1:100). After washing with PBS, slices were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZSGB-Bio, Beijing, China) at 37°C for 30 min and then stained with DAB detection system kit (ZSGB-Bio). The expression and localization of these molecules in the lung were detected under light microscopy and analyzed by image-pro plus 6.0 software.
For immunofluorescence, lung tissue sections were co-incubated overnight with antibodies F4/80 (1:1,000) with iNOS (1:100) or CD163 (1:100). After washing with PBS, sections were incubated with alexa 594-labeled goat anti-rabbit secondary antibody and alexa 488-labeled goat anti-mouse/rat secondary antibody for 1 h at 37 °C and then 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Beyotime, Shanghai, China) were added for cellular nuclear staining. Co-localization of F4/80 and iNOS or CD163 was evaluated with confocal microscopy (TCS-SP8; Leica Microsystems, Wetzlar, Germany).