Alexander Lab, Jacobs School of Medicine and Biomedical Sciences, Department of Medicine, University of Buffalo
No.
#101403
Date
07/19/2017
Antigen
Commplement Factor H
Lot Number
4390101
Method validated
Immunofluorescence
Positive Control
MRL/lpr lupus mice (14 weeks of age)
Negative Control
FH knockout MRL/lpr lupus mice
Notes
Passed. The complement factor H antibody ABIN3023097 works very well on murine kidney samples and shows the expected CFH localization and strong staining.
Validation Images
IF staining with ABIN3023097 of kidneys harvested from 14 week old MRL/lpr mice sufficient and deficient in factor H. Cryosections were fixed, stained and images acquired as described in the protocol.
Full Methods
Primary Antibody
ABIN3023097
Secondary Antibody
goat anti-rabbit rhodamine conjugated antibody (ThermoFisher Scientific, T-2769, lot 6251)
Full Protocol
Prepare kidneys of wt and complement factor H ko MRL/lpr lupus mice of 14 weeks of age.
Snap-freeze kidneys in 2-methylbutane cooled on dry ice and keep at −80°C until use.
Sections were cut at 8µm using the cryostat.
Dry sections for 30min at RT.
Hydrate sections in PBS 2x for 15min at RT.
Fix cryostat sections with 4% paraformaldehyde (PFA) for 15min at RT.
Wash sections 2x with PBS for 15min at RT.
Incubate sections with primary rabbit anti-Complement Factor H antibody (antibodies-online, ABIN3023097, 0004390101) diluted 1:100 in PBS ON at 4°C.
Wash sections 2x for 15min with PBS.
Incubate sections with secondary goat anti-rabbit rhodamine conjugated antibody (ThermoFisher Scientific, T-2769, lot 6251) diluted 1:100 in PBS for 30min at RT.
Wash sections 2x for 15min with PBS.
Incubate sections with ActinGreen 488 ReadyProbes Reagent (ThermoFisher Scientific, R37122, lot 1700569) diluted 1:100 in PBS for 30min at RT.
Wash sections 2x for 15min with PBS.
Image acquisition on a Zeiss microscope (Carl Zeiss, Oberkochen, Germany).
Experimental Notes
Format
Liquid
Buffer
PBS with 0.02 % sodium azide,50 % glycerol, pH 7.3.
Preservative
Sodium azide
Precaution of Use
This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
Avoid freeze / thaw cycles
Storage
-20 °C
Storage Comment
Store at -20°C. Avoid freeze / thaw cycles.
Cheng, Malachi, Cipi, Ma, Brush, Agbaga, Punzo: "HK2 Mediated Glycolytic Metabolism in Mouse Photoreceptors Is Not Required to Cause Late Stage Age-Related Macular Degeneration-Like Pathologies." in: Biomolecules, Vol. 11, Issue 6, (2021) (PubMed).
This gene is a member of the Regulator of Complement Activation (RCA) gene cluster and encodes a protein with twenty short consensus repeat (SCR) domains. This protein is secreted into the bloodstream and has an essential role in the regulation of complement activation, restricting this innate defense mechanism to microbial infections. Mutations in this gene have been associated with hemolytic-uremic syndrome (HUS) and chronic hypocomplementemic nephropathy. Alternate transcriptional splice variants, encoding different isoforms, have been characterized.,CFH,AHUS1,AMBP1,ARMD4,ARMS1,CFHL3,FH,FHL1,HF,HF1,HF2,HUS,Immunology & Inflammation,Cell Intrinsic Innate Immunity Signaling Pathway,CFH