Mammary gland tissues were excised immediately after euthanasia and fixed overnight in 4% paraformaldehyde (Servicebio, Wuhan, China). Tissues were paraffin-embedded and sectioned (5 μm), with sections dewaxed in xylene and rehydrated in an alcohol series, prior to staining with hematoxylin and eosin (HE; Beyotime Biotechnology Co. Ltd, Shanghai, China). Histological scoring was performed, without knowledge of treatment group, to grade tissue necrosis, dislodged epithelial cells, polymorphonuclear neutrophilic granulocyte inflammation, and lymphocytic infiltration, as described [35 (link)].
For immunohistochemistry (IHC), sections were deparaffinized and rehydrated. Sodium citrate buffer (pH = 6.0) was used for antigen retrieval. Sections were washed 3 times in PBS, incubated in PBS with 0.5% Triton X-100 (Solarbio Biotechnology Co. Ltd., Beijing, China) for 15 min and then incubated in block solution. Then sections were immunostained with primary antibodies NLRP3 (Servicebio #GB11300, 1:1000) or Keap1 (Servicebio #GB11847, 1:2000) at 4 °C overnight. Thereafter, sections were incubated with HRP-conjugated secondary antibodies (Servicebio #GB23303, 1:200) and stained with 3,3’-diaminobenzidine (DAB) (Servicebio #G1211).
For immunohistochemistry (IHC), sections were deparaffinized and rehydrated. Sodium citrate buffer (pH = 6.0) was used for antigen retrieval. Sections were washed 3 times in PBS, incubated in PBS with 0.5% Triton X-100 (Solarbio Biotechnology Co. Ltd., Beijing, China) for 15 min and then incubated in block solution. Then sections were immunostained with primary antibodies NLRP3 (Servicebio #GB11300, 1:1000) or Keap1 (Servicebio #GB11847, 1:2000) at 4 °C overnight. Thereafter, sections were incubated with HRP-conjugated secondary antibodies (Servicebio #GB23303, 1:200) and stained with 3,3’-diaminobenzidine (DAB) (Servicebio #G1211).
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