Donkey anti goat igg

We fixed iMNs with 4% paraformaldehyde and blocked them with 5% donkey serum with 0.1% Triton X-100 in 1X PBS. We incubated the cells overnight at 4 °C with the following primary antibodies: anti-SMI32 (mouse monoclonal, 1:1000, BioLegend, cat. no. SMI-32R) and anti-ISL1 (goat polyclonal, 1:250, R&D Systems, cat. no. AF1837). We subsequently rinsed the cells and incubated them with species-specific Alexa Fluor 488-conjugated secondary antibody (donkey anti-mouse immunoglobulin G (IgG), 1:1000, Life Technologies, cat. no. A-21202) and Alexa Fluor 594-conjugated secondary antibody (donkey anti-goat IgG, 1:1000, Life Technologies, cat. no. A-11058). We counterstained nuclei using DAPI (1 μg/mL). We acquired the images using Nikon/Leica microscopes with 10x magnification.

For IHC, frozen sections were washed with 1X PBS three times followed by incubating with 0.3% H₂O₂/0.3% horse serum in 1X PBS for 5 minutes at RT. Then, slides were washed in TNT buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.05% Tween-20) 3 times. Slides were then blocked one hour with TNB buffer (0.1M Tris pH 7.5, 0.15M NaCl, 0.5% blocking reagent) at RT, followed by incubation with primary antibody (COL1A1, Rockland, 1:1000) diluted in TNB buffer overnight at 4°C. Slides were then washed with TNT buffer 3 times, incubated with Horseradish peroxidase (HRP) conjugated secondary antibodies (Life Technologies) for 2 hours at RT, signals were developed by using the DAB staining kit (Vector Laboratories, Cat.# SK-4100). For IF staining, frozen sections were washed in PBS, and then blocked by 5% donkey serum in PBST for one hour at RT, followed by incubation with primary antibodies (α-SMA, 1:3000; COL1A1, Rockland, 1:1000; KLF4, Cell Signaling Technology, 1:50; FBXO32, 1:100, ECM Bioscience; PDGFRB, 1:50, Cell Signaling Technology) overnight at 4°C. Alexa Fluor 488 or 568-conjugated secondary antibodies (Donkey anti-Mouse IgG, or Donkey anti-Rabbit IgG or Donkey anti-Goat IgG; Life Technologies, 1:250 dilution) were used for the visualization of signals. Nikon DS Ri1 and SPOT software were used for the image acquisition and analysis.

Vascular tissues were harvested, washed using PBS and fixed for 6–8 h at 4 °C in 4% paraformaldehyde. Then, tissues were dehydrated in 30% sucrose solution overnight at 4 °C, embedded in OCT and frozen at − 80 °C. Frozen tissues were sliced into 10-μm thick sections using a cryostat (Leica, CM1950). Immunofluorescence staining was performed as described [27 (link)]. The primary antibodies used in this assay were αSMA antibody (Sigma, F3777, 1:500), SM22 (Abcam, ab14106, 1:200), CNN1 (Abcam, ab46794, 1:200), SMMHC (Abcam, ab53219, 1:200), RFP antibody (Rockland, 600-401-379, 1:50) and Ki67 (Abcam, ab53219, 1:200). The Alexa Fluor-conjugated secondary antibodies used in this study were Donkey anti-Mouse IgG (Invitrogen, A21202 for Alexa Fluor 488), Donkey anti-Rabbit IgG (Invitrogen, A31572 for Alexa Fluor 555), Donkey anti-Rabbit IgG (Invitrogen, A32731 for Alexa Flour 488) and Donkey anti-goat IgG (Invitrogen, A32816 for Alexa Fluor 555). Images were taken using the Nikon A1 confocal microscope. The co-staining area was quantified using the colocalization tool of Fiji. The mean gray value was calculated using the Fiji measurement tool.