Mab3402x

Day 1. Organotypic hippocampal slices were placed in 24-well plates, washed in PBS-TX, and blocked for 60 min with blocking buffer (BB, 10% normal goat serum in PBS-TX). Slices were then incubated overnight at 4 °C under slight agitation in a solution with two primary antibodies, a rabbit anti-TRPV2 antibody (1:200 in BB, product code: ab6183, Abcam, Cambridge, UK) and a mouse anti-NeuN antibody to immunostain the neurons (1:400 in BB, product code: MAB377, Millipore, Billerica, MA, USA).
Day 2. After extensive washing, the sections were incubated for 2 h at room temperature in the dark with AlexaFluor 635 goat anti-rabbit secondary antibody (1:400 in BB, product code: A31577, Thermo Fisher Scientific, Waltham, MA, USA), and then for 2 h at room temperature in the dark with AlexaFluor 555 donkey anti-mouse (1:400 in BB, product code: A31570, Thermo Fisher Scientific) and AlexaFluor 635 goat anti-rabbit (1:400 in BB). After washing, the astrocytes were immunostained using a mouse anti-GFAP antibody conjugated with the fluorochrome AlexaFluor 488 (1:500 in BB, product code: MAB3402X, Millipore, Billerica, MA, USA).

PFA-fixed L4 spinal cord segments were embedded in O.C.T. compound (Tissue-Tek, Sakura, USA), and 10-μm serial transverse sections were sliced using in a cryostat. The sections were blocked with 5% normal goat serum for 2 h and incubated overnight at 4 °C in a humidified chamber with the following primary antibodies: rabbit polyclonal anti-PDGF BB (1:100) (ab16829, Abcam), mouse monoclonal anti-NeuN (1:400) (ab104224, Abcam), mouse monoclonal anti-GFAP (1:50) (MAB3402x, Millipore), mouse monoclonal anti-OX-42 (1:100) (MCA275G, AbDsreotec), rabbit polyclonal anti-RFP (1:400) (r10367, Abcam), rabbit polyclonal anti-SP (1:100) (AB1566, Chemicon) and rabbit polyclonal anti-CGRP (1:200) (ab22560, Abcam). The primary antibodies were dissolved in 2% normal goat serum. The sections were washed 3 times with PBS for 5 min each and incubated with the appropriate secondary antibodies: goat anti-rabbit (1:400) (A-24921, Invitrogen) and goat anti-mouse (1:400) (A-24920, Invitrogen) for 2 h at 37 °C. The sections were washed with PBS 3 times for 5 min each, stained with DAPI and mounted. To confirm the specificity of the PDGF labeling, the synthetic immunogen (HangZhou HuaAn Biotechnology Co. Ltd) was pre-incubated with the PDGF-BB antibody. Then, sections in this experimental control were performed by the same process as described above (see Supplementary Fig. S1).

Immunohistochemistry was performed on paraffin-embedded mouse brain sagittal sections (5 µm) after deparaffinization and antigen retrieval. Triple immunostaining for NeuN + , GFAP + , IBA1+ cells was performed using a mouse anti-NeuN antibody for neurons (1:400; MAB377, Millipore), a rabbit anti-IBA1 antibody for total microglia (1:300; 016-20,001, WAKO) and a mouse anti-GFAP antibody conjugated with Alexa Fluor 488 (1:500; MAB3402X, Millipore) for astrocytes^{76 (link)}. Alexa Fluor 635 anti-rabbit (1:600; Alexa Fluor 555 anti mouse antibody (1:600; A31577 and A31570, Thermo Fisher Scientific) were used as secondary antibodies.