Hrp labeled goat anti rabbit igg h l

Hematoxylin and eosin (H&E) staining, immunohistochemical (IHC) staining and immunofluorescence staining were all conducted using standard techniques (Servicebio Co, China). Briefly, for the IHC, the slides were deparaffinized and antigen retrieval was then performed using a microwave oven in EDTA, pH = 8.0 (Servicebio, Wuhan, China). Primary antibodies of HIF-1α (Abcam, Rabbit IgG polyclonal, Cambridge, UK), CD31 (Servicebio, Rabbit IgG polyclonal), and NLRP3 (Servicebio, Rabbit IgG polyclonal) were applied overnight before HRP-labeled Goat Anti-rabbit IgG (H+L) (Servicebio) incubation for 50 min at room temperature. DAB was used as chromogens and slides were counterstained with hematoxylin before mounting. For the immunofluorescence staining, primary antibodies for CD31 (Servicebio, Rabbit IgG polyclonal) and α-SMA (Servicebio, mouse monoclonal 1A4) and Cy3 conjugated Goat Anti-rabbit IgG (H+L) (Servicebio) or Alexa Fluor^® 488-conjugated Goat Anti-mouse IgG (H+L) (Servicebio) were used for generating fluorescence staining. The stained sections were subjected and observed by microscopy (Olympus BX53, Tokyo, Japan). The average IOD of HIF-1α, CD31, and NLRP3 in five randomly selected areas for each group were calculated by using Imagepro Plus 6.0 (Media Cybernetics, Inc.).

Primary antibodies against PSMB5 (Abcam, Cambridge, UK) were used for IHC staining, which were incubated at 4°C overnight before being incubated for 50 min at room temperature with HRP-labeled Goat Anti-Rabbit IgG (H+L) (Servicebio). The antigen-antibody response was visualized using diaminobenzidine (DAB). Microscopy was used to photograph the slides (Olympus BX53). Image Pro Plus 6.0 was used to calculate integrated optical density (IOD) in three or five randomly chosen locations for each group (Media Cybernetics, Inc.).