UniProt Accession Number	Reagent Type	Target Name / Protein Biomarker	Target Species	Host Organism	Isotype	Clonality	Vendor	Catalog Number	Conjugate	RRID	Availability	Method	Tissue Preservation	Target Tissue	Tissue State	Detergent	Antigen Retrieval Conditions	Dye Inactivation Conditions	Recommend	Agree	Disagree	Contributor	Notes
Q58EE9	Primary Antibody	GFAP	Zebrafish	Mouse	IgG2b	SMI 25	BioLegend	837508	AF488	AB_2728555	Stock	IBEX2D Manual	4% PFA Fixed Frozen	Retina	GFP transgenic line	0.1% Triton-X-100	pH 6 (10 mM Sodium Citrate) for 20 minutes in a pressure cooker	1 mg/ml LiBH4 30 minutes + light	Yes	0009-0000-2047-4228 [1]	NA	0009-0000-2047-4228	1

Publications

1. A. Kothurkar et al., “Iterative Bleaching Extends Multiplexity (IBEX) imaging facilitates simultaneous identification of all cell types in the vertebrate retina”, bioRxiv, 2024, doi: 10.1101/2024.02.28.582563.

Additional Notes

1. Prior to IBEX imaging, antigen retrieval was performed by heating the slides for 20 minutes in 10 mM sodium citrate (pH 6). This step quenched the signal in most of the zebrafish GFP transgenic lines tested, but the GFP can be boosted in later rounds of immunolabelling using a primary antibody against GFP. Longer dye inactivation conditions reflect requirements to inactivate antibodies conjugated with FlexAble kits imaged in the same cycle with this GFAP AF488 antibody. See methods section of bioRxiv. Most AF488 conjugates dye inactivate within 15 minutes of 1 mg/ml LiBH4 (see fluorescent probes).