Alexa fluor 488 labeled goat anti rabbit igg

Rat knee joints were fixed with 4% paraformaldehyde for 48 h, decalcified with 10% EDTA (pH 7.2–7.4) for 4 weeks, then processed and embedded in paraffin. Afterwards, the samples were cut into 4 μm-thick sections and stained with safranin O–fast green (SOFG) and toluidine blue. Collagen II (Abcam, ab34712) and TRAP (Servicebio, G1050-50 T) were detected by immunohistochemistry. The expression levels of TRAF6 and CTSK were determined by immunofluorescence. Primary and secondary antibodies were used as follows: rabbit anti-Collagen II (Abcam, ab34712), rabbit anti-TRAF6 (Abcam, ab40675), rabbit anti-CTSK (Abcam, ab19027), HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303) and Alexa Fluor® 488-labeled goat anti-rabbit IgG (Servicebio, GB25303). Inverted microscope (Olympus, CKX53) equipped with a cooled CMOS camera (Tucsen, FL-20BW) was used for image acquisition. Semi-quantitative analyses of immunohistochemistry and immunofluorescence staining were performed using ImageJ software (version 1.53) as described before [35 (link)].

The following antibodies were used: anti-Bcl-2 pAb (Bioss, Proteintech Group), anti-Cytochrome C Rabbit pAb (ABclonal Technology), anti-Bax Rabbit pAb (Wanlei Biotechnology Co., Ltd), anti-Bak Rabbit pAb (ABclonal Technology), anti-caspase-3 Rabbit pAb (ABclonal Technology), anti-caspase7/cleaved-caspase7 Rabbit pAb (ABclonal Technology), IL-1β (Wanleibio Biotechnology Co., Ltd), anti-aggrecan Rabbit pAb (ABclonal Technology), Coll1A2 Rabbit pAb (ABclonal Technology), anti-GAPDH Rabbit pAb (ABclonal Technology), HRP goat anti-rabbit IgG (ABclonal Technology), Alexa Fluor® 488 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd), Cy3 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd).

The cells were fixed with 4% formaldehyde (catalog number: 10010018; Sinopharm Chemical Reagent Co., Ltd.) for 30 min and sealed with 5% bovine serum albumin (BSA) (catalog number: A8010; Solarbio). The treated cells were then incubated with 0.5% Triton X-100 (catalog number: T8210; Solarbio) for 20 min at 25°C. The cells were stained with anti-glucose-6-phosphatase catalytic subunit 2 (G6PC2) antibody (catalog number: CSB-PA873624LA01HU, 1:100; CUSABIO) overnight at 4°C and stained an hour later at 37°C in the dark with Alexa Fluor® 488-labeled goat anti-rabbit IgG (catalog number: GB25303, 1:100; Servicebio). 4′,6-Diamidino-2-phenylindole (DAPI) (catalog number: S2110; Solarbio) was used to stain the nuclei for 10 min. Images were taken using a fluorescence microscope (model number: DMIL LED; Leica).