Donkey anti rat cy3

The heart and aorta were removed and fixed in 4% PFA for 2 hours.The heart was transferred into PBS containing 30% sucrose (wt/vol) overnight at 4 °C before being immersed in OCT compound and stored at −80 °C. Eight-micrometer-thick cryosections of the aortic sinus were prepared. Cross-sections of the aortic sinus were stained with anti-LYVE-1 (ABCAM) and anti-CD68 (Biolegend) antibodies, and then incubated with the appropriate secondary antibodies. As macrophages can also be positive for LYVE-1, adventitial lymphatic capillaries were identified as LYVE-1⁺ CD68⁻ cells forming vessel-like shapes. Whole-mount immunohistochemical analysis of the ear dermis to visualize lymphatic vessels was performed as described previously47 (link). Ear dermis were stained for lymphatic capillaries (anti-LYVE-1, ABCAM) at 4 °C, and then sections were incubated with Alexa Fluor 647 conjugated donkey anti-rabbit antibody and Cy3 donkey anti-rat (Jackson ImmunoResearch). All imaging was performed on a Fluoview FV10i (Olympus). All vessel counts were performed by one observer. The relative quantification of the number of lymphatic capillaries (LYVE-1⁺ vessels), their diameter and the total surface area they occupy was determined by computer-assisted morphometric analysis. Neutral lipid assessment in atherosclerotic lesions was performed by Oil-red-O (ORO) staining (Sigma).

Retinal dissection was performed following established protocols in the Frankfort lab (Frankfort et al., 2013 (link); Tao et al., 2020b (link)). Dissected whole-mount retinas were fixed with 4% paraformaldehyde for 1 h at room temperature and blocked with 10% donkey serum overnight. Retinas were then incubated in primary antibodies [Collagen IV (EMD Millipore cat#AB756p; 1:300), CD31 (BD bioscience cat#550274; 1:50), and RBPMS (Phospho solutions cat#1832; 1:250)] diluted with 3% donkey serum for 5 days at 4°C, followed by overnight incubation at 4°C in secondary antibodies [Alexa fluor 647 donkey anti-rabbit (Jackson Immuno Research Labs cat# 711-605-152; 1:300), Cy3 donkey anti-rat (Jackson Immuno Research Labs cat#712-165-153; 1:300), Alexa fluor 488 donkey anti-guinea pig (Jackson Immuno Research Labs cat#706-545-148; 1:300), and Hoechst 33,342 nuclear staining (Invitrogen cat#H3570; 1:1,000)] diluted with 3% donkey serum.

Immunofluorescence was performed as described previously19 (link) using the following primary antibodies: rat anti-BrdU (dividing cell marker, 1:500, Thermo Scientific), mouse anti-Zpr1 (red/green cones, 1:250, ThermoFisher), mouse anti-GS (Müller glia, 1:500, Millipore), and mouse anti-HuC/D (1:300, amacrine and ganglion cells, ThermoFisher). The following secondary antibodies were used: Alexa Flour 488 donkey anti-mouse IgG (1:1000, ThermoFisher), Cy5 donkey anti-mouse (1:1000, ThermoFisher), Alexa Four 488 goat anti-rat IgG (1:1000, ThermoFisher), and Cy3 donkey anti-rat (1:1000, Jackson Immunoresearch). Antigen retrieval for BrdU staining was performed by either boiling the sections in 10 mM sodium citrate for 20 min and cooling for another 20 min or treating the sections with 2 N HCl at 37 °C for 25 min, followed by a rinse with 0.1 M sodium borate solution (pH 8.5) for 5 min.

Cryo-sections were washed 3 × 15 min with PBS and permeabilized in 0.5% TritonX-100 in PBS for 20 min, washed with PBS and blocked for one hour in blocking solution (0.1% Tween-20, 10% FCS, 0.1% BSA and 3% donkey serum). Sections were incubated with the primary antibody overnight at 4 °C. All antibodies were diluted with blocking solution. Sections were washed 4 × with PBS-0.1% Tween-20 and one time with PBS on a shaker and incubated with secondary antibodies and DAPI, diluted with blocking solution for 2 h at room temperature. After additional washing in PBST and PBS, sections were mounted with fluorescence mounting medium (DAKO, S3023), dried in the dark overnight and stored at 4 °C. Following antibodies and compounds were used: Rat monoclonal anti-F4/80 (1:100, Abcam Cat# ab6640, RRID:AB_1140040), HCS LipidTOX Green (1:400, Life technologies, catalog H34475), Cy3-Donkey anti Rat (1:800, Jackson ImmunoResearch Labs Cat# 712–165-153, RRID:AB_2340667). All images were acquired with a Leica SP8 confocal microscope using LAS AF software (Leica). Images were analyzed using ImageJ software (win64,1.53c).

The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; Cat# 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-FLAG (Cell Signaling Technology; Cat# 14793; RRID: AB_2572291; LOT# 5), rat monoclonal anti-FLAG (BioLegend; Cat# 637301; RRID: AB_1134266; LOT# B318853), rabbit polyclonal anti-SARS-CoV-2 NSP2 (GeneTex; Cat# GTX135717; RRID: AB_2909866; LOT# B318853), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from Sven Reiche (Bussmann et al., 2006 (link)), sheep polyclonal anti-SARS-CoV-2 ORF3a (Rihn et al., 2021 (link)), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; Cat# NBP3–07972; LOT# 25966–2102).
Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch (Cy3 donkey anti-rat #712–165-153, Cy3 donkey anti-mouse #715–165-151, Cy5 donkey anti-rabbit #711–175-152, Cy5 donkey anti-sheep #713–175-147), Li-Cor (IRDye 680RD donkey anti-mouse #926–68072, IRDye 680RD goat anti-rabbit #926–68071) and Invitrogen (Alexa Fluor 680 donkey anti-sheep #A21102).