Dec 09, 2024
Fluorescence immunohistochemistry
- Ashley Seifert1
- 1University of Kentucky
Protocol Citation: Ashley Seifert 2024. Fluorescence immunohistochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrmd8g5b/v1
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: December 06, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 114535
Keywords: ASAPCRN
Disclaimer
Note that any protocol involving animals should be reviewed and approved by your Institutional Animal Care and Use Committee (IACUC) before use.
Abstract
This protocol describes the procedure for immunohistochemistry detected by fluorescence used in the Seifert Lab. It assumes a starting sample of a paraffin-embedded tissue section.
Protocol materials
XyleneFisher ScientificCatalog #X3P-1GAL
100% Ethanol
100% Ethanol
Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Hoechst 33342Cell Signaling TechnologyCatalog #4082
Prolong GoldThermo Fisher ScientificCatalog #P36930
Day 1
Day 1
Deparaffinize slides to 100% EthanolContributed by users by a series of:
- 2 washes of00:05:00 each with 2X XyleneFisher ScientificCatalog #X3P-1GAL
- 2 washes of 00:02:00 each with 100% EthanolContributed by users
Rehydrate to ddH2O by a series of:
- 00:03:00 wash with 90% Ethanol
- 00:01:00 wash with 70% Ethanol
- 2 washes of 00:01:00 each with ddH2O
(SKIP IF FROZEN)
Perform retrieval, optimized for antigen and treatment
Note
For a first run, can try heat + citrate buffer pH 6, heat + DAKO high pH 9, proteinase K
*After heat, transfer directly to ddH2O to cool before proceeding to first wash with TBS
**For matrix proteins, try 2' proteinase K
Wash for 00:05:00 with Tris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
Block for at least 00:30:00 at Room temperature in 15 μL/mL appropriate serum in TBS
Note
The serum is typically whatever the secondary antibody was raised in; for multiple labeling, use a 50:50 mixture of the two sera
(SKIP IF NOT AMPLIFYING SECONDARY ANTIBODY)
Avidin/Biotin Block
Block with avidin for 00:15:00 at Room temperature
Wash for 00:05:00 in TBS
Block with biotin for 00:15:00 at Room temperature
Wash for 00:05:00 in TBS
Incubate with primary antibody or control in 15 μL/mL serum in TBS Overnight at 4 °C in a humidity box
Note
The preferred control is IgG at the same concentration as the primary antibody, but otherwise just
leave out the primary antibody
Day 2
Day 2
Wash for 00:05:00 in TBS
Incubate with secondary antibody (biotinylated secondary to the species of the primary) at 1:400 in 15 μL/mL serum in TBS for 00:30:00 at Room temperature
Note
1:400 is usually a good dilution, but this can be reduced if background is too high
Wash for 00:05:00 in TBS
Incubate with desired AlexaFluor-conjugated secondary antibody at 1:400 in TBS (without serum) for 00:30:00 at Room temperature
Wash for 00:05:00 in TBS
Wash for 00:05:00 in ddH2O
Incubate with 1 μg/mL Hoechst 33342Cell Signaling TechnologyCatalog #4082 for 00:05:00 at Room temperature
Wash with ddH2O
Stand slide at an angle and allow to drip-dry for a few minutes
Cover slip
Note
If using Prolong GoldThermo Fisher ScientificCatalog #P36930 can cover slip when a bit wet