Donkey anti rabbit igg alexa 647

Fixed embryos were cryoprotected in 20% sucrose overnight at 4°C. Then, the embryos were embedded in 20% sucrose:TissueTek OCT (Sakura, Alphen aan den Rijn, South Holland, Netherlands), sectioned in a cryostat (Leica, CM1850 UV), and collected on gelatin-coated slides. The cryosections were dried, washed with PBS and blocked with 3% NGS (Normal Goat Serum, Jackson Immunoresearch, West Grove, PA, United States) in PBST (PBS with Triton X-100 0.2%, Sigma) for 1h at room temperature in a humid chamber. The primary antibody was diluted in blocking solution and incubated overnight in a humid chamber at room temperature. The primary antibodies used were anti-DsRed2 mouse (1:50; Santa Cruz Biotechnology, cat. Sc-101526), rabbit anti-GFP IgG (1:200; Molecular Probes, cat. A-6455) and mouse anti-HNK-1 IgM (DSHB, cat. 3H5). The secondary antibodies were incubated for 2 h at room temperature and in a humid chamber. The secondary antibodies used were goat anti-mouse IgM coupled to Alexa 488 (1:200, Molecular Probes, cat. A-21042), goat anti-rabbit IgG -Alexa 488 (1:200, Molecular Probes, cat. A-11008), donkey anti-rabbit IgG - Alexa 647 (1:800, Molecular Probes, cat. A31573), goat anti-rabbit IgG–Alexa 568 (1:500, Molecular Probes, cat. A11011) and goat anti-mouse IgG–Alexa 568 (1:500, Molecular Probes, cat. A11004). Finally, the slides were washed and mounted in FluoroShield with DAPI.

Untreated lungs of Col-GFP mice were fixed with 4% PFA overnight at 4 °C and then inflated with low-melting point agarose. 100 μm sections were made using a vibratome VT1000S (Leica). The sections were cleared using a CUBIC method as described previously^{46 (link)}. After delipidation with Reagent-1A (10 wt% triton, 5 wt% NNNN-tetrakis (2-HP) ethylenediamine, 10 wt% urea, 25 mM NaCl), the sections were stained with anti-α-SMA-alexa 647 (R&D), or anti-collagen 4 (LSL) followed by donkey anti-rabbit IgG-alexa 647 (Thermo Fisher). The sections were then treated with refractive index-matching reagent (Reagent-2; 25 wt% urea, 50 wt% sucrose, 10 wt% triethanolamine) and imaged using W1 spinning disk confocal microscope (Nikon). Images were processed using Image J version 1.52i. 3D-reconstruction of z-stack images was performed using Icy version 2.0. For whole lung imaging after transfer, 4% PFA-fixed lungs were cleared with Reagent-1A and treated with Reagent-2, followed by imaging for GFP signal using Nikon AZ100 microscope configured as light sheet microscopy. Autofluorescence signal in RFP channel was used to visualize the lung structure. Maximum projection images were generated using Image J.