Goat anti rabbit secondary antibody

Prepared paraffin-embedded sections were deparaffinized, rehydrated, retrieved and blocked with 5% BSA (w/v) in PBS at ambient temperature for 30 min. Then, sections were incubated with 100 μM biotin labeled RP-1 peptide or human specific rabbit anti-CD44 monoclonal antibody (1:200, ZSGB-BIO, Beijing, China) overnight at 4°C. After PBS washing for three times, sections were subsequently incubated with streptavidin-HRP (1:500, Proteintech, Wuhan, China) and goat anti-rabbit secondary antibody (1:300, BOSTER, Wuhan, China). Diaminobenzidine (DAB, ZSGB-BIO, Beijing, China) was used as a chromogen. Finally, the sections were counterstained with hematoxylin and mounted with neutral balsam.

The immunohistochemical method [22 (link)] was used to detect PGC-1α, Bax, cytochrome C, and Bcl-2 expression. Brain paraffin sections were dewaxed and hydrated with xylene and ethanol, and then incubated with a 0.3% hydrogen peroxide solution for 15 min. The sections were placed in citric acid buffer (pH6.0), and the antigen was repaired using a microwave. The sections were blocked with 5% BSA at 37 °C for 30 min, then the sections were incubated with antibodies for PGC-1α (1:500), Bax (1:200), cytochrome C (1:150), and Bcl-2 (1:300) at 4 °C overnight. Next, the sections were incubated with goat anti-rabbit secondary antibody (BOSTER Biological Technology Co., Ltd., Wuhan, China) at 37 °C for 30 min. Then, the sections were stained using a DAB Staining Kit (BOSTER Biological Technology Co., Ltd., Wuhan, China), and then stained with hematoxylin. The positive cells were brown under a light microscope. From each group, three sections were randomly selected and three fields from each section were selected (400×). The average optical density of positive cells was observed, and the expression of PGC-1α, Bax, cytochrome C, and Bcl-2 was analyzed by IPP software.

On days 7 and 14 after operation, total proteins were extracted from the L4–5 spinal cord segment of the rats. Separating glue and stacking glue separately were prepared. Afterward, the stacking glue was solidified, and the comb was pulled out. A 5-μl molecular marker was added to the first well, and then 10 μl of samples was added to the subsequent wells in the order of the groups, and then electrophoresis and transfer film were performed. After the membrane transfer was completed, the membranes were placed in 5% skimmed milk powder for 90 min. Subsequently, the membranes were placed in the P2 × 4R rabbit polyclonal antibody (1:1,000, Millipore, Bedford, MA, United States), NF-_KB p65 rabbit polyclonal antibody (1:1,000, Sigma-Aldrich), Nf-_kB p-p65 (1:1,000, Sigma-Aldrich), or rabbit β-actin polyclonal antibodies (1:3,000, Boster Biological Technology, Ltd.) and incubated overnight in a 4°C refrigerator. They were placed in goat anti-rabbit secondary antibody (1:5,000, Boster Biological Technology, Ltd.) and incubated for 90 min, and then washed three times with TBST for 10 min each time. Subsequently, the chemiluminescence reaction was carried out. Image Pro Plus version 6.0 image analysis software was used to perform the grayscale analysis of the protein bands. The relative band strength of the target protein was normalized with internal parameters (β-actin).

Western blot was performed to quantify the expression of SLC17A9 and ChAT. The methods were as described in the previous article. The membranes were incubated overnight at 4 °C with rabbit anti-SLC17A9 (1:1000; MBL), sleep anti-ChAT (1:1000; Abcam), and rabbit anti-actin (1:1000; Abcam; 1:1000; CST). Goat anti-rabbit secondary antibody (1:5000, BOSTER) and Donkey anti-goat secondary antibody (1:2000, Servicebio) in 5% non-fat milk were used to incubate for 1 h at room temperature.

Paraffin-embedded tissues were cut into 5-μm-thick slices for IHC examination. The sections were incubated with anti-p-Akt (1:200) and anti-p-Bad (1:200) antibodies (both from Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. Then, they were incubated with goat anti-rabbit secondary antibody (Boster, Wuhan, China), and visualized with a 3,3-diaminobenzidine (DAB) kit (Boster, Wuhan, China). Finally, the mounted sections were observed and photographed under a microscope at 200× magnification (DM2500, Leica, Solms, Germany).

Paraffin-embedded normal and tumor tissues were dewaxed, dehydrated, antigenically fixed, and sliced into 4 μm-thick sections for immunohistochemical staining. The sections were incubated with anti-human TREM2 antibody (Clone #237,920, 1:500, R&D) and anti-human CD206 antibody (#24,595, 1:1000, Cell Signaling Technology) overnight at 4 °C, followed by incubation with goat anti-rabbit secondary antibody (BOSTER), and finally stained with DAB (BOSTER) for visualization. DAPI (Abmole, USA) was used for staining of cell nuclei and blocking of sections prior to observation.

Total protein was extracted using RIPA buffer supplemented with a protease inhibitor cocktail (NCM Biotech Co. Ltd., China) on ice. Protein concentration was determined using the BCA assay (Boster Biological Technology Co. Ltd, China). Cell lysates were denatured in sodium dodecyl sulfate (SDS) sample loading buffer at 95 °C for 10 min and separated on 10 % polyacrylamide gels. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked with 5 % non-fat milk in TBST buffer for 1 h at room temperature and then incubated overnight at 4 °C with rabbit polyclonal anti-PDIA3 (1:2000, Proteintech, USA), rabbit polyclonal anti-AKT (1:2000, Proteintech, USA), rabbit anti-phospho-AKT (Ser473) (1:1000, Proteintech, USA), or rabbit polyclonal anti-β-actin (1:5000, Boster Biological Technology Co. Ltd, China). The membranes were then incubated with goat anti-rabbit secondary antibody (1:5000, Boster Biological Technology Co. Ltd., China) for 2 h at room temperature. Bands were visualized using enhanced chemiluminescence (ECL; NCM Biotech, USA), imaged, and quantified using a ChemiDoc Imaging System (Bio-Rad, USA).