Aug 08, 2019
Version 2
UC Davis - Immunohistochemistry BODIPY V.2
- Jennifer Rutkowsky1
- 1University of California, Davis
- Mouse Metabolic Phenotyping CentersTech. support email: info@mmpc.org
Protocol Citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry BODIPY. protocols.io https://dx.doi.org/10.17504/protocols.io.56rg9d6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 05, 2019
Last Modified: August 08, 2019
Protocol Integer ID: 26545
Keywords: Immunohistochemistry BODIPY (493/503), Diabetes, cardiovascular disease, cognitive impairment
Abstract
Summary:
BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids. It has uses for identifying and quantifying lipid droplets in various cell and tissue type. Abnormal lipid droplet accumulation can be associated with various metabolic disorders such as Diabetes, cardiovascular disease and cognitive impairment.
Materials
MATERIALS
ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6148
Prolong gold (with dapi)Life TechnologiesCatalog #P36930 (P36931)
Xylene
Ethanol
Tri-sodium citrate
BODIPY™ 493/503Life TechnologiesCatalog #D3922
0.1M Phosphate Buffered Saline pH 7.4
coverslip CorningCatalog #2935-245
Reagent Preparation:
Reagent1: Block/Perm
Reagents and Materials:
BSA
Saponin
0.1M Phosphate buffered saline (PBS), pH 7.4
Procedure:
For 100mL combine 1g BSA and 0.05g Saponin in 100mL PBS
Reagent 2: 4% Paraformaldehyde
Reagents and Materials:
Paraformaldehyde
0.1M Phosphate buffered saline (PBS), pH 7.4
Procedure:
For 100mL combine 4g Paraformaldehyde 95mL PBS. In a fume hood, heat solution to 55C on a heat plate while stirring. Add 2-3 drops of concentrated HCl to bring into solution. Cool to room temperature and adjust pH to 7.4. Bring up to 100 mL and then sterile filter with 0.22µM filter.
Reagent 3: Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Reagents and Materials:
Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Procedure:
Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.
Note:
Sigma-Aldrich RRID:SCR_008988
Life Technologies, RRID:SCR_008817
Safety warnings
WARNING:
Paraformaldehyde is, toxic, flammable and considered a carcinogen
Xylene, ethanol and methanol are all flammable and should be used in fume hood away from open flames or sparks
All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions established by CDC when handling and disposing of infectious agents.
Frozen tissue sections (10µM) on slides (tissues embedded in OCT) are then fixed in 4% paraformaldehyde for 5 minutes.
a. Alternatively paraffin embedded sections are deparaffinized through 3 100% xylene 3 min wash
b. Sections were rehydrated through 100%, 95%, 70%, 50%, and 30% then into deionized water (3min each).
c. Antigens are unmasked by steaming in Sodium citrate for 10 min.
Wash three times for ten minute in PBS
The sections where then incubated in 0.1mg/ml BODIPY® 492/515 in DMSO for 30 minutes in the dark.
Wash three times for ten minute in PBS
Coverslip mounted with Prolong gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI).