Alexa fluor 488 affinipure donkey anti mouse igg h l

LA tissues from mice were fixed with 4% phosphate-buffered formalin for 24 h, paraffin embedded, serial sectioned at 5μM thick, deparaffinized, stained with Masson’s trichrome staining kit (HT15 Trichrome Staining Kit; Sigma-Aldrich), sealed and stored at 4°C. Immunofluorescence was performed on deparaffinized sections using primary antibodies: vimentin (Abcam, ab8978, 1:200), CDKN2A/p16INK4a (Abcam, ab211542, 1:200) and p21 (Abcam, ab188224, 1:200). Then, the sections were incubated with secondary antibodies Alexa Fluor 594-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Yeason, 34212ES60, 1:300) or Alexa Fluor 488-AffiniPure Donkey Anti-Mouse IgG (H+L) (Yeason, 34106ES60, 1:300). DAPI was used to stain the nuclei.

For cellular immunofluorescence analysis, primary cultured AFs were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% TritonX-100 in PBS for 10 min. After blocking with 5% BSA for 1 h at room temperature, the cells were incubated with mouse antibody against H3K27me3 (ABclonal, A16199, 1:200), vimentin (Abcam, ab45939, 1:200), CDKN2A/p16INK4a (Abcam, ab211542, 1:200) and p21 (Abcam, ab188224, 1:200) at 4 °C overnight, rabbit normal IgG (Abcam, ab6702) was used as negative control. The cells were then incubated with Alexa Fluor 488-AffiniPure Donkey Anti-Mouse IgG (H+L) (Yeason, 34106ES60, 1:300), Alexa Fluor 594-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Yeason, 34212ES60, 1:300) and DAPI at room temperature for 1 h.