A8010

Macrophages were washed with PBS three times. Four percent fixative solution (Solarbio, P1110) was added to the Petri dish for 10 min. Then, the solution was removed and cells washed three times. Next, 0.5% Triton X-100 (Solarbio, T8200) was added to the dish for 10 min. The solution was removed and cells washed three times. Five percent BSA (Solarbio, A8010) was added to the dish, and it was incubated for 1 h at room temperature. Then, primary antibodies were added to the M0 (GLRX1: 1:500, Abcam, ab45953; CD11b: 1:100, Proteintech, 66519-1-lg) and M2 macrophages (GLRX1: 1:500, Abcam, ab45953; CD163: 1:100, Abcam, ab156769) (18 (link)), respectively, and they were incubated overnight at 4°C. The solution was removed and cells washed three times. Secondary antibodies (DyLight 488 goat antirabbit polyclonal antibody, Abcam, ab96899, 1:200; DyLight 594 goat antimouse polyclonal antibody, Abcam, ab96881, 1:200) were used for 1 h at room temperature. The solution was removed and cells washed three times. Prolong™ Diamond Antifade Mountant with DAPI (Invitrogen, P36962) was added to the dish, and photos were taken with confocal microscopy.

Using the same method as above, the brain tissue of young rats was fixed, embedded, sliced, dewaxed, and rehydrated. After treatment with 3% H₂O₂ methanol solution for 12 min, the slices were immersed in citrate buffer (pH 6.0, M053201, Mreda, Beijing, China) at 95 °C for 10 min and cooled. Subsequently, they were blocked with bovine serum albumin (5% BSA, A8010, Solarbio, Beijing, China) for 20 min. Polyclonal primary antibodies, comprising rabbit anti-human BDNF antibody (1:300, orb10181, Biorbyt, Wuhan, China) and rabbit anti-human CPEB2 antibody (1:300, orb182706, Biorbyt, Wuhan, China), were added and reacted for one night at 4 °C. After being washed with PBS 3 times for 5 min each, the sections were incubated at room temperature with Alexa Fluor 546 goat anti-rabbit IgG secondary antibody (1:1000, A-11010, Thermo Fisher, Shanghai, China) diluted with PBS. Then, hematoxylin stain was applied for 2 min, and alcohol hydrochloride was applied for 2 s for differentiation. Using the same method as above, the slices were dehydrated with ethanol, treated with xylene, sealed with neutral gum, and observed under a light microscope (Revolbe FL, Echolaboratories, USA). A corresponding area was selected on each slice and analyzed with ImageJ (version 5; National Institutes of Health) under the same conditions.

For Immunofluorescence staining, paraffin-embedded tissue sections were subjected to a heat-mediated antigen retrieval procedure. To be specific, after sections were dried, dewaxed, and hydrated, they were heated to repair antigens. Antigens repair was performed with citric acid buffer (PH6.0) in a 92 °C water bath for 60 min and then restored at room temperature. Following that, tissue sections were incubated overnight with a primary antibody [(Col-2 pAb, #28,459–1-AP, Proteintech, Wuhan, China), (Sox-9 Rabbit mAb, #82,630, Cell Signaling Technology, MA, USA)] at 4 °C. The secondary antibody was then added to the sections, which were incubated for 60 min. The nucleus was counterstained with 75% DAPI (C0060, Solarbio) for 10 min. Finally, anti-fluorescence quencher was applied to seal the sections.
Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA (A8010, Solarbio). The primary antibodies were then incubated overnight in PBS at 4˚C, followed by an hour of incubation with the secondary antibodies at room temperature. The rest of the steps are the same as above.

The cultured hAMSCs and SCLCs were first digested in a 0.05% trypsin (Solarbio, T1300) solution and then re-suspended in pre-cooled PBS (Solarbio, P1010) containing 0.04% BSA (Solarbio, A8010). One sample for each group. We prepared a single-cell library, following the manufacturer’s instructions (Chromium Single Cell 3′ v2 Reagent Kit; 10X Genomics, Pleasanton, CA, United States). Briefly, the cell suspension (2,000 cells/μl) was loaded on to Chromium microfluidic chips. The mRNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed using reagents from the kit according to the manufacturer’s instructions. Sequencing (NovaSeq) was performed by Novogene. RNA-seq data used in this study were deposited into the GEO database (GSE161066).