Rabbit anti myc tag

Rats were perfused with 4% paraformaldehyde (PFA), tissues dissected and postfixed at 4°C for 24 hours in 4% PFA. The brain tissue was then cryoprotected in a 30% sucrose (Thermo Fisher) in PBS. The tissue was then sectioned on a cryostat (Leica CM 3050S) into free-floating sections at 20 μm thickness, which were preserved in PBS with 0.1% w/v sodium azide at 4°C. To stain the tissue slices, they were first washed 3 times in PBS and then put in blocking buffer containing 3% horse serum, 0.1% bovine serum albumin (BSA), and 1% Tween-20 in 1X PBS. Subsequently, incubation of primary antibodies took place for 16 hours at 4°C. Primary antibodies are as follows: 1 : 100 rabbit anti-myc-tag (OriGene), 1 : 200 anti-NeuN (Millipore), and 1 : 250 WFA (PNN marker, Sigma-Aldrich). After another three washes with PBS-T, incubation of appropriate secondary antibody at a concentration of 1 : 500 mixed with 1 : 10000 Hoechst (Thermo Fisher) in 1X PBS took place for one hour at RT. After three washes with 1X Tris-sodium chloride buffer (TNS), the slices were mounted on slides (twin frost glass slides, Thermo Scientific). To preserve the signal, FluorSave Reagent (Calbiochem) was applied to all slices and left to dry for 24 hours.

For immunohistochemistry, virus injection, neuronal cultures and Western Blots, the following are used: goat anti-Nptx2 (Santa Cruz, sc-12125), chicken anti-NeuN (Millipore, abn91), rabbit anti-myc-tag (OriGene, TA100029), rabbit anti-parvalbumin (Abcam, ab11427), biotinylated WFA (Sigma-Aldrich, L1516), mouse anti-6X his-tag (Abcam, ab18184), Hoechst (Thermo Fisher, H3570), his-tag NPTX2 protein (R&D Systems, 10889), chABC (Sigma-Aldrich, C3667), test hyase (Sigma-Aldrich, H3506), strep hyase (Sigma-Aldrich, H1136), and lenti-cmv-Nptx2 lentivirus (10⁷ TU/mL, OriGene, MR206833L1V).