Environmentally friendly transparent dewaxing liquid

After antigen retrieval (see in Immuno uorescence), spinal cord tissues were incubated in 3% H 2 O 2 . Then, sections were blocked with 3%BSA (BioFroxx) at room temperature for 1 hour. The slices were incubated with rat anti-C3 (1:20, Abcam, UK) primary antibodies at 4℃ overnight. The next day, after washed with PBS 3 times, goat anti-rat secondary antibody (1:200, ZSGB-Bio) was used for incubating at room temperature for 30 minutes and stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB, ZSGB-Bio). Next, sections were stained with hematoxylin (Solarbio) and differentiated with alcohol-HCl. After rinsing with tap water, slices were dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol solution in turns, and made transparent with Environmentally friendly transparent dewaxing liquid (Solarbio). Then sealed with neutral gum and dried. Images were obtained with an upright uorescence microscope (Olympus, Tokyo, Japan).

For astrocytes, the cells planted in a 24-well plate were washed with PBS 3 times, treated with 4% paraformaldehyde (Solarbio) for 15 min, 0.5% Triton X-100 penetrated for 10 min, and then blocked with 10% normal goat serum (Solarbio) for 1 hour. Then, cells were incubated with primary antibody at 4℃ overnight. The next day, samples were incubated with secondary antibodies for 30min at room temperature. Finally, Antifade Mounting Medium with DAPI (Beyotime Biotechnology) was used for sealing.
Spinal cord tissues were sectioned after para n embedding. After treated with Environmentally friendly transparent dewaxing liquid (Solarbio) for depara nizing, slices were hydrated in 100%, 95%, 90%, 80%, 70% ethanol solution in turns. Then, sections were heated in citrate buffer for 15min for antigen retrieval.
The procedure of staining was the same as that was performed in cells.
An upright uorescence microscope (Olympus, Tokyo, Japan) was used for obtaining images.