Immunohistochemistry (Paraffin)
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- Xylene.
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Wash Buffer:
- 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1 L 1X TBST, add
100 ml 10X
Tris Buffered Saline with Tween® 20 (TBST)(#9997) to
900 ml
dH20, mix.
- 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 mL 10X Wash Buffer,
Phosphate Buffered Saline(#12528)
to
900 ml
dH20, mix.
- SignalStain® Antibody Diluent: (#8112)
- 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of
SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 ml of
dH2O.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml
dH2O.
- Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
- TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
- 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking
Solution (5X) (#15019).
- Prolong® Gold AntiFade Reagent(#9071),
Prolong® Gold AntiFade Reagent with DAPI(#8961).
- (optional) TrueBlack® Lipofuscin Autofluorescence Quencher (#92401).
B. Deparaffinization/Rehydration
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 minutes each.
- Incubate sections in two washes of 100% ethanol for 10 minutes each.
- Incubate sections in two washes of 95% ethanol for 10 minutes each.
- Wash sections twice in dH2O for 5 minutes each.
C. Antigen Unmasking
- Heat slides submersed in 1X EDTA unmasking solution in a microwave until boiling is initiated; follow
with 15 min at sub-boiling temperature (95°-98°). No cooling is
necessary.
D. Staining
- Wash sections in dH2O three times for 5 minutes each.
- Incubate sections in 1X TBST for 5 min.
- Block each section with 100-400 µl of preferred blocking solution for 1 hour at room
temperature.
- Remove blocking solution and add 100-400 µl primary antibody diluted in
SignalStain®
Antibody
Diluent to each section.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each protected from light.
NOTE: See below for
optional
TrueBlack® Lipofuscin Autofluorescence Quencher protocol.
- Coverslip slides with Prolong® Gold Antifade Reagent or Prolong® Gold
Antifade
Reagent with DAPI.
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store
slides flat at 4°C protected from light.
TrueBlack® Lipofuscin Autofluorescence Quencher protocol
Following Section D Step 6:
IMPORTANT: TrueBlack® Lipofuscin Autofluorescence Quencher is not compatible with
detergent. Any steps
involving detergent must be done before applying TrueBlack® Lipofuscin Autofluorescence
Quencher.
- Prepare TrueBlack® Lipofuscin Autofluorescence Quencher solution by diluting 1:20 in
70% ethanol. Vortex to mix.
NOTE: Quenching solution should be made fresh prior to use and discarded if precipitate is
visible. We recommend heating the vial of stock solution of TrueBlack® Lipofuscin
Autofluorescence
Quencher, 20X in DMF to 70°C prior to dilution in order to avoid precipitate
formation.
- Immediately cover tissue sections with 100 µL - 200 µL of quenching solution for 30
seconds at room temperature.
IMPORTANT: Do not allow sections to dry out. Sections may tolerate longer incubations (up to
3 minutes) so long as they remain hydrated.
- Tap slides on an absorbent towel to collect excess TrueBlack® Lipofuscin
Autofluorescence Quencher
before transferring to 1X PBS.
- Rinse three times in 1X PBS for 5 min each.
- Proceed with counterstaining/mounting.
Protocol Id: 2924
Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies
A. Solutions and Reagents
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow
Cytometry Kit (Methanol) #13593, or
individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
- 4% Formaldehyde, Methanol-Free (#47746)
- 100% Methanol (#13604): Chill before use
- Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by
dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
B. Fixation
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
- Pellet cells by centrifugation and remove supernatant.
- Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
- Fix for 15 min at room temperature (20-25°C).
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
- Alternatively, cells may be stored overnight at 4°C in 1X PBS.
C. Permeabilization
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Permeabilize for a minimum of 10 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
D. Immunostaining
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate
waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
- Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.
posted July 2009
revised June 2020