Goat anti rat alexa 568

Frozen tissue was cryosectioned on an OTF 5000 cryostat (Bright) at 10-μm thickness through the muscle length. Transverse sections were fixed in ice-cold acetone and blocked in 1% BSA, 1% goat serum, 0.1% Triton X-100, and 1× PBS. Sections subsequently were incubated with rat anti-laminin antibody (1:1,000, Sigma) at 4°C overnight. Slides were washed three times in 1× PBS and 0.05% Tween-20 prior to a 1-hr incubation with goat anti-rat Alexa568 (1:1,000, Life Technologies). Dystrophin was stained using Mouse-on-Mouse Basic kits (Vector Laboratories) following the manufacturer’s instructions. Monoclonal mouse anti-dystrophin 6C5 (1:50, Novocastra Laboratories) and goat anti-mouse Alexa488 (1:1,000, Life Technologies) were used. An additional 15-min staining with 1 μg/mL DAPI (Sigma) was performed prior to mounting in Mowiol 4-88 (Sigma).

Fly brains were dissected in PBS and fixed in 4% PFA with 0.1% TritonX for 25 minutes. Brains were washed in 0.3% PBT and incubated first with primary (24-72h) and then secondary (24-48h) antibodies in 0.3% PBT supplemented with 5% NGS at 4°C. Brains were mounted in Vectashield mounting medium (Vector Laboratories) and imaged on Leica TCS SP5 laser-scanning confocal microscope. We used the following antibodies: rabbit anti-GFP (Torrey Pines, 1:400), mouse anti-nc82 (DSHB, deposited by E. Buchner, 1:200), rat anti-RFP, (Chromotek 5F8, 1:50), mouse anti-ChAT (DSHB, deposited by P. Salvaterra, 1:50), goat anti-rabbit Alexa 488 (Thermo Fisher Scientific, 1:200), goat anti-rat Alexa 568 (Thermo Fisher Scientific, 1:200) and goat anti-mouse Alexa 647 (Thermo Fisher Scientific, 1:200).

Double immunofluorescent staining was performed using the following pairs of antibodies: H11 (α1) and Mab5 (α5) for amphibians and H43 and Mab5 for shark. Secondary antibodies were goat anti-rat-Alexa568 (red) and goat anti-mouse-Alexa488 (green) (Thermo Fisher Scientific).

CldU and yH2AX stainings were performed as previously described (Benedict et al, 2018 (link)). Shortly, cells were cultured on cover slides, labelled with 100 μM CldU for 30 min, washed with PBS + Ca²⁺/Mg²⁺ and fixed in 70% EtOH for 10 min. Subsequently, cells were treated with MeOH for 5 min and permeabilized with 1.5 M HCl for 20 min. Cells were blocked using PBS + Ca²⁺/Mg²⁺, 0.5% Tween, 0.25% BSA and 5% FCS for 30 min. The cells were incubated with the primary antibodies rat-anti BrdU (Clone BU1/75, 1:20 dilution; Abcam) and mouse-monoclonal phosphorylated H2AX (Upstate, 1:100 dilutions) for 2 h and washed with PBS + Ca²⁺/Mg²⁺ and 0.5% Tween. Next, the cells were incubated with the secondary antibodies goat–anti Rat Alexa 568 (1:100 dilution; Invitrogen) and Goat-antimouse Alexa 488 (1:100 dilution; Invitrogen). DNA was stained using Topro3. Images were made on a confocal Leica SP5 system using a ×63 oil objective with LAS-AF software and analyzed using a customized Macro on ImageJ software.

Following primary antibodies were used for Immunofluorescence (IF) and Western blotting (WB): rabbit anti-GFP (1:200 IF, Invitrogen, A11122), chicken anti-GFP (1:200 IF, Aves, GFP-1020), rabbit anti-GFAP (1:100 IF, Dako, Z0334), mouse anti-NeuN (1:50 IF, Millipore, MAB377), rat anti-L1 (1:100 IF, Millipore, MAB5272), rabbit anti-Calretinin (1:500 IF, Millipore, MAB5054), goat anti-Nrp1 (2 µg/ml for function blocking, R and D, AF566), goat anti-VEGFR2 (1:15 IF, R and D, AF644), rabbit anti-VEGFR2 (1:1000 WB, Cell Signaling, 2479), rabbit anti-(phospho)VEGFR2 (Y1175) (1:500 WB, Cell Signaling, 2478), rabbit anti-(phospho/SFK (Y416) (1:200 IF, Invitrogen, 44660G), mouse anti-beta-III-tubulin (1:150 IF, Sigma, T5076), goat anti-VE-Cadherin (1:1000 WB, R and D, AF1002), Phalloidin (1:400 IF, Sigma, P1951). The following secondary antibodies were used: donkey anti-rabbit Alexa488 (Jackson Immunoresearch, 711-545-152), donkey anti-rabbit Alexa568 (Life Technologies, A10042), donkey anti-goat Alexa568 (Molecular Probes, A11057), goat anti-mouse Alexa568 (Invitrogen, A11031), goat anti-rat Alexa568 (Invitrogen, A11077), goat anti-chicken Alexa488 (Molecular Probes, A11039), donkey anti-goat HRP (Jackson Immunoresearch, 705–0350147), donkey anti-rabbit HRP (Jackson Immunoresearch, 711-035-152), donkey anti-mouse HRP (Jackson Immunoresearch, 715-035-150).

Differentiated WT or FcRn KO podocytes were fixed in 4% paraformaldehyde. After fixation, podocytes were rinsed with PBS, permeabilized in 0.1% Triton-X100 in PBS (PBS-X), blocked in 5% BSA in PBS-X and incubated overnight at 4°C with the following primary antibodies depending on the experiment: rat anti-mouse LAMP1 (1:500, Santa Cruz, cat. # sc-19992), goat anti-mouse IgG (1: 100, Vector Labs, cat. #NC0207995), rat anti-mouse IgG1 (1:100, Biolegend, cat. # 50169164), rabbit anti-mouse Rab11 (1:200, Cell Signaling, cat. # 5589), rabbit anti-mouse Rab7 (1:200, Cell Signaling, cat. #9367). Podocytes were rinsed and incubated with the appropriate secondary antibodies (Alexa 568 goat anti-rat, Alexa 488 goat anti-rat, Alexa 488 goat anti-rabbit, Alexa 568 donkey anti-goat, Alexa 568 goat anti-rabbit, Invitrogen). Hoechst was used to stain nuclei and Alexa 635 phalloidin was used to stain actin. Images were acquired using a Zeiss LSM 780 microscope. Colocalization between immune complexes and LAMP1 or ICs and Rab11 was evaluated using the Coloc2 Tool in ImageJ (NIH). Lysosomal area was also assessed using ImageJ.