Alexa fluor 488 conjugated anti rabbit antibody

Because the AM was too thin to be analyzed, three sheets of AM were layered and embedded using the Tissue-Tek O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan). The specimens were cut to a thickness of 7 μm and placed on Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific, Waltham, MA, USA). After they dried for 30 minutes at room temperature, the samples were fixed on a slide for 5 minutes with 95% EtOH. Hematoxylin–eosin (H&E) staining was performed, followed by observation using optical microscopy.
Collagen type IV, a constituent protein of the basement membrane, was identified by immunohistochemistry. The tissues attached to the slides were washed with PBS and treated with a blocking solution containing 1% bovine serum albumin for 1 hour to inhibit any non-specific reactions. The samples were incubated overnight with a primary anti-collagen type IV antibody (1:200; Abcam, Cambridge, UK) at 4°C. After washing, Alexa Fluor 488 conjugated anti-rabbit antibody (1:200; Abcam) was used as a secondary antibody for 1 hour at room temperature. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole.

Cell suspensions from lumbar spinal cords were prepared as described above, except for the fixation step. Fresh tissue was processed and blocked for 30 min with 2% normal mouse serum containing an anti–CD16/CD32 antibody (BD Bioscience; FcγR blocker) and then stained with a mix of 1:50 PE-Cy7–conjugated CD11b antibody (BioLegend; RRID: AB_312799), 1:50 rabbit anti-mouse TMEM119 antibody (Abcam; RRID:AB_2744673), and 1:50 PerCP-Cy5.5–conjugated CD24 antibody (BioLegend; RRID:AB_1595491); cells were then washed and incubated with (1:200) secondary Alexa Fluor 488–conjugated anti-rabbit antibody (Abcam; RRID:AB_2630356) and incubated for 30 min on ice. After that, cells were washed and incubated with 1:50 Alexa Fluor 647–conjugated Glast1 antibody (Novus Biologicals) and 1:100 dilution of Life/Death Ghost Red 780 dye (Cell Signaling) for 30 min on ice. Cells were washed with sorting buffer and filtered before being sorted into lysis buffer using a FACS-Aria cell sorter (BD Biosciences). Three technical replicates, each with 400 cells from the same animal, were sorted. See Fig. S2, A and B for sorting strategy and analysis of purity of sorted microglia.