Basic IHC Protocol - Floating Sections
*Please read “Notes” prior to using this protocol for the first time.
Solution recipes are on the page 2!
- Remove sections from storage solution into PBS, if necessary.
- Transfer sections to blocking solution in a 6 well plate and incubate with gentle agitation for 1 hour at room temp. (Prepare primary antibodies in Carrier Solution, 500µl per section.)
- Using a 12-well plate, add antibodies to appropriate wells, then transfer sections into wells. Use carrier solution for secondary controls and blank section.
- Incubate overnight at 4°C with gentle agitation.
- Fill wells of a 6-well plate with 10ml of Wash solution. Transfer each section to a separate well and wash 5min, 15min, 5min with agitation. (Transfer to new wells for each wash. (Prepare secondary antibodies in Carrier Solution, 500µl per section.)
- Using a 12-well plate, add antibodies to appropriate wells, then transfer sections. Use carrier solution for blank sections.
- Incubate for 1hour at room temp with gentle agitation, protected from light.
- Fill wells of a 6-well plate with 10ml of Wash solution. Transfer each section to a separate well and wash 5min, 15min. Transfer to new wells for each wash.
- Transfer sections to new well containing PBS and wash 5min.
- DAPI staining (optional):
- Prepare a 12 well plate with 500µl 300nM DAPI per well, one well.for each section.
- Transfer first section out of PBS and into DAPI. Start a 2min timer
- Transfer remaining sections to DAPI wells.
- After 2min incubation, move sections back to the wells with PBS for one rinse.
- Mount sections onto gelatin-coated slides and place coverslip using MOWIOL.
Solutions for Section Staining
Wash Solution: Makes 0.5L
To 435ml of dH2O, add:
50ml 10X PBS, pH 7.4
7.5ml 20% Triton (0.3% Final)
5ml Serum that matches host of secondary antibody (1% Final)
Bring to 500ml with dH2O.
Adjust pH to 7.3 – 7.4.
Carrier Solution: Makes 100ml
To 100ml of Wash Solution, add:
0.55g BSA (0.5% Final)
Blocking Solution: Makes 50ml
10ml serum that matches host of secondary antibody
40ml Carrier Solution
NOTES: This protocol should be viewed as a guide – the steps can be modified according to need! Please check your cell and antibody datasheets for specific conditions that may influence the steps you include, exclude or alter.
- Volumes and well sizes indicated are for individual rodent sections. Larger sections or sheets of sections will require more volume to cover the sample and larger dishes to incubate and wash. Floating sections are easier to handle if they do not bunch up during incubation steps.
- Protein crosslinking due to fixation may mask some antigens. Antigen retrieval requirements are usually indicated in your antibody datasheets or in previously published data. Such steps would occur prior to blocking.
- Handle DAPI with care – it has mutagenic properties. DAPI wastes, including washes, should be collected separately as chemical wastes.
- Mounting floating sections can be done a number of ways:
- Draw a PAP Pen circle on a slide, then transfer a section with a small amount of PBS into the circle. Pipette away the excess solution until the section sits flat on the glass. Multiple sections can be mounted in multiple circles on one slide.
- Fill a large glass petri dish with PBS. Move one section to the dish and unfold it, if necessary. Place a slide into the PBS at an angle, submersing nearly half of the slide in the solution. Using a glass hook or small brush, tease the section onto the glass, holding it in place while the solution runs down into the dish. Make fine adjustments to the placement with a brush, then blot away any excess PBS. Multiple sections can be mounted down the length of the slide in this manner.
In either case, allow excess solution to evaporate and section to bind to the glass, then apply mounting medium and coverglass. Store flat and dark overnight to set mounting medium. Sections are generally stable at room temp (dark) while imaging.
Transfer to 4°C for longer storage.