*IMPORTANT: Please refer to the product webpage to determine whether a product is validated and approved for use with frozen tissue sections. Please see product webpage for appropriate antibody dilution and unmasking solution.
NOTE: Please see product webpage for product-specific protocol recommendations.
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%).
- Hematoxylin (optional).
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Fixative Options: For optimal fixative, please refer to the product datasheet.
- 10% neutral buffered formalin.
- Acetone.
- Methanol.
- 3% formaldehyde: To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
- 10X Tris Buffered Saline (TBS) Wash Buffer: (#12498) To prepare 1 L 1X TBS add 100 ml of 10X TBS to 900 ml dH2O, mix.
- Methanol/Peroxidase: To prepare, add 10 ml 30% H2O2 to 90 ml methanol. Store at -20°C.
- Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton X-100 to 9.5 ml 1X TBS.
- Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114).
- Substrate: SignalStain® DAB Substrate Kit (#8059).
B. Sectioning
- For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
- Section tissue at a range of 6–8 µm and place on positively charged slides.
- Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides).
C. Fixation Options
NOTE: Consult product datasheet to determine the optimal fixative.
- After sections have dried on the slide, fix in optimal fixative as directed below.
- 10% Neutral buffered formalin: 10 min at room temperature. Proceed with staining procedure immediately (Section D).
- Cold acetone: 10 min at -20°C. Air dry. Proceed with staining immediately (Section D).
- Methanol: 10 min at -20°C. Proceed with staining immediately (Section D).
- 3% Formaldehyde: 15 min at room temperature. Proceed with staining immediately (Section D).
- 3% Formaldehyde/methanol: 15 min at room temperature in 3% formaldehyde, followed by 5 min in methanol at -20°C (do not rinse in between). Proceed with staining immediately (Section D).
D. Staining
- Wash sections in wash buffer two times for 5 min.
- Incubate for 10 min at room temperature in methanol/peroxidase.
- Wash sections in wash buffer two times for 5 min.
- Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution to each section.
- Incubate overnight at 4°C.
- Equilibrate SignalStain® Boost Detection Reagent to room temperature.
- Remove antibody solution and wash sections in wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
- Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount sections with coverslips.
posted February 2010
revised November 2013