Rat anti cd68

Brain tissue was collected at P15 as previously described. Sections were permeabilized with PBS and 0.5% Triton X-100 for 30 min, and blocked with PBS, 0.3% Triton, and 5% goat serum for 30 min at room temperature. CD68 was immunodetected by ON incubation at 4 °C with primary antibodies (rat anti-CD68 1:500, Serotec) followed by secondary antibodies (goat anti-rat A647, 1:600, Life Technologies) incubation in PBS with 0.3% Triton and 10% goat serum at 4 °C ON. Secondary dendrites of bright GFP + neurons were imaged in medial stratum radiatum of CA1 using Leica SP5 confocal resonant scanner microscope with a × 63/1.4 oil-immersion objective, at a lateral pixel size of 40 nm and an axial step of 130 nm. Images were deconvolved using Huygens software (40 iterations, 0.1 of quality change, theoretical point spread function) and sharpened using Image J software (NIH). Interactions were determined after 3D visualization in Imaris as follows: appositions were considered when 20–50% of the spine head surface was covered by microglia, encapsulation when > 70% was covered.

The following primary antibodies were used in this study: anti-FLAG (M2) and anti-myc (9E10) from Sigma-Aldrich, anti-GAPDH from Abcam (ab8245), anti-LC3B (GTX127375) and anti-C9orf72 (GTX119776) from GeneTex, anti-Cathepsin D (sc-6486), anti-Ulk1 (sc-33182) and anti-C9orf72 (sc-138763) from Santa Cruz Biotechnology, anti-C9orf72 (AP12928b) and anti-FIP200 (17250-1-AP) from Proteintech, anti-SMCR8 from Bethyl Laboratories, anti-WDR41 from Abgent, rat anti-CD68 from AbD Serotec, sheep anti-progranulin from R&D systems and anti-mouse LAMP1 (553792) from BD Biosciences. Anti-mouse prosaposin antibody was generated by Pocono Rabbit Farm and Laboratory and was previously characterized [55 (link)]. Anti-C9orf72-long isoform [52 (link)] was a gift from Dr. Janice Robertson (University of Toronto); anti-GFP antibody was a gift from Dr. Anthony Bretscher (Cornell University); and anti-GPP130 was a gift from Dr. William Brown (Cornell University). The following secondary antibodies were used: donkey anti-mouse 800 and donkey anti-rabbit 800 from LI-COR, AlexaFluor donkey anti-goat 680, donkey anti-rabbit 680, donkey anti-mouse 680, and donkey anti-rat 680 from Invitrogen, and donkey anti-mouse 568 from Biotium. Hoechst stain was obtained from Invitrogen. Brefeldin A (BFA) and nocodazole were obtained from Sigma-Aldrich and used at a final concentration of 300 μM and 20 mM, respectively.

Immunostaining was carried out using previously established protocols [20 (link)]. The sections were incubated in a blocking solution (5 % normal donkey serum, 2 % BSA, and 0.1 % Triton X-100) for 1 h at room temperature (RT). The sections were then incubated overnight at 4 °C with goat anti-Arginase-1 (1:50; Santa Cruz Biotechnology, CA, USA) and rat anti-CD68 (1:100; AbD Serotec, Oxford, UK). The sections were incubated for 1 h at RT with Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and then mounted on gelatin-coated slides and cover-slipped with VectaShield medium (Vector Labs, Burlingame, CA, USA). All images were acquired using confocal laser scanning microscopy (LSM700; Carl Zeiss, Germany).

Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91^phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Brain sections were blocked with 10% goat serum in PBS containing 0.2% Triton X-100 and then immunostained with microglial marker mouse anti-CD11b (1:200, Abcam, Cambridge, UK), rabbit anti-Iba1 (1:400, Wako Chemical), rat anti-CD68 (1:200, AbD Serotec) and astrocyte marker mouse anti-GFAP (1:200, Merck Millipore) in blocking solution at 4 °C overnight. Omission of the primary antibody incubation step and/or isotype-matched control antibodies was used to ascertain staining specificity. Brain sections were then incubated at room temperature for 1 hour with AlexaFluor goat anti-mouse IgG-488, goat anti-rabbit IgG-488 and goat anti-rat IgG-555 (Molecular Probes, Life Technologies, USA) at a dilution of 1:200. Brain sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole, 1:2000, Sigma) and mounted using Prolong Gold (Molecular Probes).