Nov 15, 2019
IHC Fluorescent Frozen Sections
- 1East Tennessee State University
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Elizabeth Smith 2019. IHC Fluorescent Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.7ayhifw
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: September 12, 2019
Last Modified: November 15, 2019
Protocol Integer ID: 27704
Abstract
Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures.
Attachments
Guidelines
Frozen tissues cut in 30 micron intervals were used in this protocol. Do not touch the tissue on the slide or it will come off.
Materials
MATERIALS
Normal Donkey SerumCatalog #017-000-121
Citifluor AF-1 anti-fading solutionElectron Microscopy Sciences
KimWipesFischer Scientific
Triton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #93426
PBS
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
Superfrost™ Disposable Microscope Slides, White; 3 x 1 in. x 1mmThermo FisherCatalog #12550123
Bovine Serum AlbuminThermo FisherCatalog #15561020
Coplin Staining JarThermo FisherCatalog #194
Scientific Device Humidity/Slide Moisture ChamberThermo FisherCatalog #23769522
Slides boxes wrapped in tinfoil to store slides in -20 degrees Celsius prior to cutting.
Various primaries and secondaries dependent upon structure of interest.
Glass coverslips in various sizes depending on tissue size.
Primaries:
Neuropeptide Y (NPY) Antibody from Immunostar Catalog#22940
Anti-Tyrosine Hydroxylase (TH) from Millipore Catalog#AB1542
VAChT from Synaptic Systems Catalog#139 103
Anti-PGP9.5 antibody from abcam Catalog#ab108986
Secondaries:
Alexa Fluor 488 conjugated AffiniPure Donkey Anti-Rabbit IgG from Jackson ImmunoResearch Catalog#711-545-152
Alexa Fluor 555 conjugated Donkey Anti-Goat IgG from ThermoFisher Catalog#A-21432
Alexa Fluor 555 conjugated Donkey Anti-Rabbit IgG from ThermoFisher Catalog#A-31572
Alexa Fluor 594 conjugated Donkey Anti-Rabbit IgG from Jackson ImmunoResearch Catalog#711-585-152
Alexa Fluor 594 conjugated Donkey Anti-Goat IgG from ThermoFisher Catalog#A-11058
Day 1: Using the PAP Pen, carefully draw a water barrier circle around the tissue sections on the slide – allow this circle to dry for several seconds or up to approx. one minute
Rinse slides with PBS (pH 7.3-7.4): 4 x 5 min each
Rinse slides with 0.5% BSA + 0.4% Triton X-100 in PBS): 1 x 10 min
Remove slides one at a time and using a clean Kimwipe, carefully wipe around the tissue sections to dry the slide
Place the slides into a black, covered slide incubation box/humidity box
Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
Pour off the blocking buffer
Replace with primary antibody solution (antibody of choice diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
Incubate tissue with primary antibody overnight in incubation box
Day 2: Rinse slides with PBS: 4 x 5 min
Rinse slides with 0.5% BSA + 0.4% Triton X-100 + PBS: 1 x 10 min
Place the slides into a black, covered slide incubation box/humidity box
Cover the tissue sections with blocking buffer (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS)
Allow the sections to remain in blocking buffer for 1.5-2 hrs. at RT
Prepare fluorescent secondary antibody (secondary antibody should be diluted in 1.0% BSA + 0.4% Triton X-100 + PBS)
Cover the tissue with the secondary antibody solution and incubate for 2 hrs. at RT in the incubation box. *From this point on, use low light and/or cover tissues.*
Rinse slides with PBS: 4 x 5 min
Remove excess PBS with a Kimwipe
Carefully add a drop of mounting medium to the center of the tissue and apply cover glass
Seal cover glass with clear nail polish. For thicker tissue, add a weight before sealing