3 3 diaminobenzidine dab color developing solution

The fibroblasts were seeded on coverslips prepared in 6-well plates. After 12 hours of culture, cells grew on the glass coverslips. Then, 4% paraformaldehyde (Servicebio, Wuhan, China) was added to the 6-well plates for cell fixation. For cell immunochemistry, drops of 3% bovine serum albumin (BSA) were added onto the coverslips and kept at room temperature for 30 minutes. The liquid was discarded. The cells were then incubated with the diluted primary antibodies at 4 ℃ for more than 10 hours and with the secondary antibody (G1213, Servicebio) for 1 hour at room temperature. The primary antibodies included anti-Cytokeratin antibody (GB11197, Servicebio) and anti-Vimentin antibody [5741, Cell Signaling Technology (CST), Danvers, MA, USA]. After being washed 3 times in PBS, slides were stained with 3,3’-diaminobenzidine (DAB) color developing solution (Servicebio) and then with hematoxylin stain solution (Servicebio) to counterstain the nucleus. Images were observed under a light microscope (Olympus, Tokyo, Japan).

Fresh tissues were fixed, embedded in paraffin wax, and subjected to H&E staining or IHC staining according to the established protocols. The sections were dewaxed in xylene and rehydrated in ethanol, and endogenous peroxidase was blocked by methanol containing 0.3% hydrogen peroxide for 30 minutes. For antigen retrieval, the sections were placed in plastic jars containing a citric acid antigen retrieval buffer (Servicebio, Wuhan, China) and then heated in a microwave oven. Non-specific protein binding was inhibited by treatment with normal goat serum (for rabbit polyclonal antibodies) (Servicebio) or 3% bovine serum albumin (BSA; Servicebio) for 30 minutes. Sections were incubated at 4 ℃ overnight with primary antibodies, and the tissues were covered with a secondary antibody (HRP-labeled; Servicebio) from the corresponding species of primary antibody and incubated at room temperature. The sections were then reacted with a 3,3'-diaminobenzidine (DAB) color-developing solution (Servicebio, China), counterstained with hematoxylin, and mounted. The primary antibodies used were as follows: MEF2D (Proteintech, China), PI3K (Proteintech, China), p-AKT (Proteintech, China), and BCL2 (Proteintech, China).