Donkey anti rabbit 555

Antibody staining for brains was performed according to standard protocols [27] (link). Primary antibodies used were chicken anti-GFP (1∶1,000, Invitrogen), rabbit anti-RFP (1∶1,000, Invitrogen), mouse anti-nc82 (1∶1,000, DSHB), Guinea pig anti-Dati (1∶1,000, gift from T. Isshiki [35] (link)), rat anti-Elav (1∶1,000, DSHB), and mouse anti-FasII (1∶100, DSHB). Secondary antibodies used were donkey anti-mouse 647 (1∶500), goat anti-rat 555 (1∶500, Invitrogen), donkey anti-guinea pig 647 (1∶500, Invitrogen), donkey anti-chicken 488 (1∶500, DyLight), and donkey anti-rabbit 555 (1∶500, Invitrogen). All samples were mounted in SlowFade (Invitrogen) and scanned on a Zeiss LSM 700 confocal microscope. Images generated from Z-stacks taken at 1 or 2 µm intervals are displayed as maximum intensity projections using Zeiss Zen 2009 or as orthogonal projections/surface projections using Image J.

When necessary, depending on the tissue penetrance and antigen recognition ability of the antibody used, antigen retrieval was performed by microwaving slides in 10 mM sodium citrate buffer for 1 min at maximum power, followed by 10 min at minimum power. Slides were then washed in 1× PBS and incubated in blocking solution (5% normal donkey or normal goat serum, 0.2% Triton^® X-100 in PBS) for 1 h at room temperature. This was followed by incubation in primary antibody overnight at room temperature. Slides were washed in 1× PBS and incubated with secondary antibody solution for 1 h at room temperature. Slides were mounted in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were as follows: rabbit anti-oligodendrocyte transcription factor 2 (1:300, AB9610, Millipore, Burlington, MA), rabbit anti-phosphorylated histone 3 (1:500, 06-570, Millipore), rat anti-somatostatin (1:50, MAB354, Millipore), rabbit anti-parvalbumin (1:1000, PV25, Swant, Marly, Switzerland), rabbit anti-calretinin (1:1000, Swant, 769913), and rabbit anti-Tbr1 (1:1000, gift from the Hevner laboratory, University of Washington School of Medicine, Seattle, WA). The following secondary antibodies were used: (1:250 dilution, Thermo Fisher Scientific): donkey anti-rabbit 555 (A31572), goat anti-rabbit 546 (A11035), goat anti-rabbit 488 (A11008) and goat anti-rat 488 (A11006).

Coronal sections were air-dried, washed with 1X PBS, blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% triton-X100 for 1 h, incubated with a primary antibody diluted in blocking solution overnight at room temperature (RT), and then washed with 1X PBS six times 10 min each and incubated with appropriate secondary antibody solution (1:250 dilution) for 1 h at RT. Slides were then washed with 1X PBS and then mounted with media-containing DAPI counterstain (SouthernBiotech, Birmingham, AL, USA). Antibodies used are goat anti-EphA4 at 1:100 dilution (R&D systems, Minneapolis, MN, USA), rabbit anti-TMEM119 at 1:100 dilution (Abcam, Waltham, MA, USA), rabbit anti-CCR2 at 1:100 dilution (Abcam, Waltham, MA, USA), rat anti-CD68 at 1:100 dilution (Thermofisher, USA), rabbit anti-Iba1 at 1:200 dilution (FUJIFILM Wako Chemicals U.S.A. Corporation), donkey anti-goat 594 (Thermofisher, USA), donkey anti-rabbit 647 (Thermofisher, USA), donkey anti-rabbit 555 (Thermofisher, USA), and donkey anti-rat 647 (Thermofisher, USA). Image acquisition was performed using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

After dissection, Drosophila midguts were fixed in 4% paraformaldehyde in 1X PBS for 45 to 90 minutes and successively washed 3 times with 0.1% TritonX in PBS. Guts to be immunostained were then incubated for an hour in blocking solution (1% bovine serum albumin, 1% normal donkey serum, and 0.1% Triton X-100 in PBS). Overnight primary antibody staining was performed at RT. Guts were washed 3 times with 0.1% TritonX in PBS and ≥2 hour secondary antibody staining was performed in PBS. Primary antibodies used: rabbit anti-pH3 (1:000, EMD Millipore), rabbit anti-β-Galactosidase (1:1000, MP Biomedicals), and mouse anti-Prospero (1:100, DSHB). Secondary antibodies used: donkey anti-rabbit-555 (1:2000, Thermo Fisher), donkey anti-mouse-488 (1:2000, Thermo Fisher), and donkey anti-mouse-647 (1:1000, Thermo Fisher). DNA was stained in 1:50,000 DAPI (Sigma-Aldrich) in PBS and 0.1% TritonX for 30min, and samples received a final three washes in PBS before mounting in antifade medium (Citifluor AF1). Imaging was performed on a Zeiss LSM 700 fluorescent/confocal inverted microscope.

FFPE slices were deparaffined and pretreated with 0.1% glycine in H₂O for 10 min at room temperature (RT). Following a 70% formic acid treatment (4 min, RT), slices were rinsed in H₂O. Slices were immerged in citrate buffer (0.01 M, pH = 6) and placed in a cooker for 20 min at 95 °C. Slices were rinsed in H₂O and treated overnight at 4 °C in 0.1 M PBS-1% BSA-0.3% Triton X-100 with the following antibodies: goat anti-IBA1 (Ab48004, Abcam, 1/300) and rabbit anti-TSPO (Ab109497, Abcam, 1/300). Slices were rinsed in 0.1 M PBS (3 × 10 min) and treated with the secondary antibodies (donkey anti-goat 488 and donkey anti-rabbit 555, Thermofisher, 1/300) in 0.1 M PBS-1% BSA-0.3% Triton X-100 for 90 min at RT. After rinsing in 0.1 M PBS (3 × 10 min), slices were mounted in Fluor save. The Axio Imager.Z2 Basis LSM 800 microscope (Zeiss) was used to take pictures at CA4 (stack of images every 0.3 µm). A pixel-based analysis in the different channels on each Z-position was realized to calculate the % of colocalization (average of average of three distinct measures per individual) using imageJ (v1.53c). The representative image shows the maximum intensity projection (Z-stack Processing, ImageJ).