Donkey anti goat 488

Eyes were dissected to obtain eyecups that were cryoprotected and frozen. Serial transversal slices (12 μm thick) were performed with cryostat (CL3050 S, Leica), then immersed in permeabilization and saturation buffer during 1 hour at room temperature, incubated overnight with 1/400 Iba1 goat (Invitrogen, Carlsbad, CA, USA; AB5076) and 1/500 GFAP rabbit (Dako, Glostrup, Denmark; Z0334) antibody solutions and revealed using Donkey anti‐goat 488 and anti‐rabbit 594 antibodies (Thermo Fisher Scientific; A11055). Images were obtained using confocal microscopy (Inverted Confocal Olympus) at ×40 magnification.

Brain tissues of patients with ICH and ICH mice were fixed in 4% paraformaldehyde and embedded in paraffin. Brain sections (5 µm thick) were made and rehydrated in a series of ethanol dilutions. Brain sections were permeabilized and incubated in blocking solution (5% goat or donkey serum in PBS solution), followed by incubation with primary antibodies: goat anti-Iba1 (Wako, Richmond, VA, USA) and rabbit anti-PBR (Abcam, Cambridge, MA, USA) at 4°C overnight. After triple washings with PBS, the slices were incubated with secondary antibodies: donkey anti-rabbit 594 (Thermo Fisher Scientific) and donkey anti-goat 488 (Thermo Fisher Scientific). After triple washings with PBS, the brain sections were stained with DAPI (Abcam). For TUNEL staining, the Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling kit (Roche, Indianapolis, IN, USA) was used according to the manufacturer’s protocol. Images were captured with a microscope (Model BX-61; Olympus, Center Valley, PA, USA), and data were analyzed with Image J (NIH). For cell counting, positively stained cells were counted in 3 comparable, randomly selected microscopic fields. The numbers of cells from 9 locations per mouse (3 fields per section × 3 sections per mouse) were averaged and expressed as positive cells per field.

Perfused fixed brains were cryopreserved in 25% sucrose and then embedded in OCT and serial cryosectioned. Infarct volume (mm³) was assessed by the Cavalieri Estimator probe using the StereoInvestigator software (MicroBrightField, Williston, VT, USA), as previously described. Briefly, six serial coronal sections, cut at 30 μm, were stained using a 0.2% Cresyl violet solution (Electron Microscopy Science, Hatfield, PA) or anti-mouse IgG-488 (Invitrogen, Waltham, MA). The total volume of infarct was quantified by estimating the area of tissue loss in the ipsilateral cortical hemisphere using six, serial coronal sections. A 100 μm spaced grid was placed over the ipsilateral hemisphere in the Cavalieri probe and infarcted area scored. The coronal sections were used for immunohistochemical (IHC) staining of CD31. Sections were blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO) in 0.2% Triton-X100, incubated with goat anti-CD31 (1 : 100, R&D systems, Minneapolis, MN, USA) overnight at room temperature (RT), washed with 1X PBS, and then incubated with donkey anti-goat 488 (1 : 250, Thermofisher, USA) for 1 hour at RT. Slides were then mounted with DAPI counterstain (SouthernBiotech, Birmingham, AL), and images were acquired using Olympus fluorescence microscope. Microvessel diameters were measured using ImageJ.

At Day 120 and 160, organoids were fixed with 4% paraformaldehyde for 30–60 min at room temperature and equilibrated to 15% sucrose in PBS, followed by 30% sucrose. Organoids were cryopreserved in 50:50 solution of 30% sucrose / PBS: tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA) and cryosectioned (15 μm). Sections were blocked with 5% normal donkey serum, 3% bovine serum albumin, and 0.1% triton-x and stained overnight with the following primary antibodies: OTX2 (R&D Systems; Cat# AF1979), Recoverin (EMD Millipore, Burlington, MA; Cat# AB5585), NRL (R&D Systems; Cat# AF2945), ARR3 (Lifespan Biosciences, Seattle, WA; Cat# LS-C368677), ML-Opsin (EMD Millipore; Cat# AB5405) and NR2E3 (R&D Systems; Cat# PP-H7223-00). The following secondary antibodies (Thermo Fisher Scientific) were incubated for 1 h: donkey anti-goat 488 (Cat# R37118), donkey anti-mouse 488 (Cat# A21202), and donkey anti-rabbit 647 (Cat# A31573). Cell nuclei were counterstained using DAPI (Thermo Fisher Scientific; Cat# 62,248). Images were acquired using a Leica TCS SPE upright confocal microscope system (Leica Microsystems, Wetzlar, Germany).

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).