Anti pkcδ

The protein levels were quantified by western blotting analysis of cell extracts using antibodies below: anti-GAPDH (1:2000, Santa Cruz, USA), anti-PN-1, anti-E-cadherin, anti-Vimentin, anti-Snail, anti-ZEB-1, anti-MMP9, anti-Oct4, anti-Sox2, anti-Nanog, anti-EGFR, anti-ERK1/2, anti-P-ERK1/2, anti-P-PKCδ, anti-PKCδ, anti-EGR1, anti-htrA1, and anti-EGF (1:1000, Abcam, USA). ImageJ software was used for protein bands analysis. Immunofluorescence assay was performed as described elsewhere^{15 (link)}, in which the monoclonal antibodies, including anti- PKCδ (1:100, Abcam) and anti-Vimentin (1:200, Abcam) antibodies were used. Cell images were captured using a laser scanning confocal microscopy (Olympus, Japan).

The expression of collagen II, collagen X and PKC was detected using immunohistochemistry. The slices were dewaxed in xylene, dehydrated in graded ethanol and incubated in 3% H₂O₂ at 37°C for 10 minutes. Next, they were washed in PBS for 5 minutes thrice, boiled in 0.01 M citric acid buffer for antigen retrieval (95°C, 15‐20 minutes) and blocked in goat serum for 10 minutes at 37°C. Slices were then incubated with primary antibody (anti‐Collagen II (1:200), anti‐Collagen X (1:50), anti‐PKC‐ε (1:50) and anti‐PKC‐δ (1:1000), Abcam, USA) at 4°C overnight and with biotin‐labelled secondary antibody (Bioworld) for 30 minutes at 37°C. Slices were counterstained with haematoxylin and observed under a light microscope.

The protocol was similar to that explained previously (Yin et al., 2020 (link)). The mice were anesthetized and consecutively intracardially perfused with ice-cold saline for 3 min and 4% PFA for 4 min. Brain tissue was extracted and post-fixed overnight in 4% PFA at 4°C, and then immersed in 30% (W/V) sucrose at 4°C until they settled. Frozen brains were sectioned into transverse slices (40 μm) with a cryostat (Leica CM1860, Wetzlar, Germany). Brain slices were used for immunofluorescence. The slices were incubated in primary antibodies, including anti-ASIC1a (1:500, mouse, Abcam, Cambridge, United Kingdom), anti-PKCδ (1:250, rabbit, Abcam, Cambridge, United Kingdom), anti-SOM (1:100, mouse, Santa Cruz, Texas, United States) and anti-GABA (1:500, rabbit, Sigma, MO, United States), at 4°C for 24 h. Then, the slices were washed with PBS three times (5 min each time) and incubated with the secondary antibodies (1:500, Invitrogen, MA, United States) for 2 h in a dark place at room temperature. The slices were observed with a Zeiss LSM880 microscope (Oberkochen, Germany) to visualize the fluorescence signals.