Donkey anti goat a21447

Hypoxia was assessed using pimonidazole HCl (Hypoxyprobe) according to manufacturer’s instructions. Ninety minutes prior to euthanasia, mice were given an intraperitoneal injection of (60 mg/kg) pimonidazole HCl diluted in sterile 1X PBS. Mice were then transcardially perfused with 20 mL saline solution and 20 mL 4% paraformaldehyde. Eyes were harvested and incubated in 4% paraformaldehyde for 30 min. Eyes were stored in 1X PBS until dissection. Hypoxia was detected using an antibody against pimonidazole (Table 1).Table 1

Summary of antibodies.

	Concentration	Company
Primary antibody (catalog number)
Rabbit anti-cCASP3 (AF835)	1:1000	R&D Systems
Goat anti-CD31 (AF3628)	1:1000	R&D Systems
Goat anti-ChAT (AB144P)	1:200	Millipore
Mouse anti-JUN (BDB610327)	1:250	Fisher Scientific
Rabbit anti-pimonidazole (PAb2627AP)	1:200	Hypoxyprobe
Rabbit anti-pJUN (9261S)	1:250	Cell Signaling
Rabbit anti-RBPMS (GTX118619)	1:250	GeneTex
Guinea pig anti-RBPMS (1832-RBPMS)	1:250	PhosphoSolutions
Goat anti-SOX2 (sc-17320)	1:200	Santa Cruz
Secondary antibody (catalog number)
Donkey anti-rabbit (A31572, A-21206)	1:1000	ThermoFisher
Donkey anti-guinea pig (706–605–148)	1:1000	Jackson ImmunoResearch
Donkey anti-goat (A21447)	1:1000	ThermoFisher
Donkey anti-mouse (A31570)	1:1000	ThermoFisher

For cell surface staining, cells were incubated in 0.5% PBS‐BSA (Sigma‐Aldrich) containing antibodies for 30 min at 4°C. Single‐cell measurements were performed on a FACS Canto flow cytometer (BD Biosciences) and FlowJo V10 software (TreeStar) was used to analyze the data. The antibody clones used are: CD86 (2331 (FUN‐1), BD Pharmingen), CD80 (L307.4, BD Pharmingen), CD83 (HB15e, BD Pharmingen), ACE2 (AF933, R&D Systems), goat‐IgG (AB‐2535864, ThermoFisher Scientific), donkey‐anti‐goat (A‐21447, ThermoFisher Scientific). For each experiment, live cells were gated on FSC and SSC and analyzed further with the markers mentioned (Supporting information Fig. S1). The authors adhered to the guidelines for the use of flow cytometry and cell sorting in immunological studies [37 (link)].