Ar1177

Slides were then incubated with specific primary antibodies against a-SMA (rat, Ab5694, 1 : 200, Abcam, USA) and Nrf2 (Ab62352, 1 : 200, Abcam, USA) at 4°C overnight. Next day, after washing with 1x PBS for 5 min/3times, slides were incubated with secondary antibody (1 : 500, Boster, China) for 1 h at room temperature. Then, samples were stained with DAPI (AR1177, BOSTER, China) and sealed in antifade fluorescence mounting medium (AR1109, BOSTER, China) with coverslips. The detection of a-SMA and Nrf2 proteins was performed under a fluorescence microscope (IX71, Olympus, Japan). Nrf-2 positive cells were counted using Image pro plus software. The percentage of Nrf-2 in the nucleus was quantitatively analyzed by the ratio of nuclear Nrf-2 positive cells to total cells in each field.

The distribution of HSF1 was assessed by immunofluorescence as described previously [26 (link)]. Briefly, the cells were fixed with 4% paraformaldehyde (G1101-500ML, Servicebio, Wuhan, China) for 15 min, permeabilized with 1% Triton X-100 (G5060-100ML, Servicebio, Wuhan, China) for 10 min, and incubated overnight at 4 °C with anti-HSF1 primary antibody (A13765, 1:100, Abclonal, Wuhan, China). Next, the cells were incubated with Cy3-labeled goat anti-rabbit antibody (BA1032, Boster, Wuhan, China) for 1 h and DAPI (AR1177, Boster, Wuhan, China) for 15 min at room temperature. The distribution of HSF1 was then observed by fluorescence microscopy (IX73-A12FL/PH, OLYMPUS, Japan).

Immunofluorescence staining was performed in vitro on coverslip cultures or frozen brain tissue sections as previously described [27 (link)]. Briefly, cells were fixed in 4% PFA for 20 min (frozen tissue sections did not require this step), washed with phosphate-buffered saline (PBS), and then blocked by QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, P0260) for 30 min at room temperature. The primary antibodies were added and incubated overnight at 4°C. Primary antibodies included mouse anti-Nestin (Millipore, MAB5326, 1 : 1000), rabbit anti-PAX6 (BioLegend, 901301, 1 : 1000), goat anti-SOX1 (R&D Systems, AF3369, 1 : 1000), rat anti-HOXB4 (DSHB, RRID: AB-2119288, 1 : 100), rabbit anti-Foxg1 (Abcam, Ab18259, 1 : 500), mouse anti-NKX2.1 (Millipore, MAB5460, 1 : 500), rabbit anti-GABA (Sigma-Aldrich, A2052, 1 : 1000), goat anti-CHAT (Millipore, AB144P, 1 : 300), mouse anti-neuronal class III β-tubulin (Tuj1; BioLegend, 801201, 1 : 2000), mouse anti-microtubule-associated protein-2 (Map2; Sigma-Aldrich, M1406, 1 : 1000), mouse anti-human nuclei (HuNu; Millipore, MAB1281, 1 : 500), goat anti-mCherry (Biorbyt, RRID: AB-2687829), and rabbit anti-NeuN (Millipore, ABN78, 1 : 1000). The corresponding fluorescent-conjugated secondary antibodies were applied for 1 h, and the nuclei were stained with DAPI (Boster, AR1177).