Basic IF Protocol  - Frozen Sections on Slides

*Please read “Notes” prior to using this protocol for the first time.

Solution recipes are on the page 2!

1. Remove slides from the freezer and allow to warm to room temp for 15min.
2. Using a PAP Pen, draw circles around sections.
3. Set slides in a staining box and apply blocking solution to each slide. Incubate at room temp for 1 hr. (Prepare antibodies in Carrier Solution)
4. Draw off excess blocking solution and apply prepared primary antibodies. Incubate overnight at 4°C in a closed, humidified box.
5. Discard antibody solution and rinse each slide with a gentle stream of PBS to remove excess antibody.  Transfer slide to a Coplin jar containing 50ml of Wash solution.
6. Wash slides 5min, 15min, 5min with agitation.  Use fresh Wash solution each time. (Prepare secondary antibodies in Carrier Solution)
7. Remove one slide at a time from Coplin jar, wiping excess solution from back of slide and around hydrophobic circle. 
8. Set slides in a staining box and apply prepared secondary antibodies to each slide.Incubate for 1hour at room temp.
9. Discard antibody solution and rinse each slide with a gentle stream of PBS to remove excess antibody.  Transfer slide to a Coplin jar containing 50ml of PBS.
10. Wash slides for 5min, 15min, 5min in PBS.
11. DAPI staining (optional): 
    • Remove one slide at a time from PBS.  Shake off excess and dry the back.
    • Add 300nM DAPI to cover each section and start a 2min timer.
    • Repeat with each slide.
    • After 2min incubation, rinse each slide thoroughly in PBS.
12. Mount coverslips 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.

• 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.
• Avoid incubating different antibodies in separate hydrophobic circles on the same slide. The high protein content of the incubation solution CAN overcome the barrier, especially if too much solution has been added to the circle.  If a slide has multiple sections, divide them into two sets:  1) Single or Dual Primary Anitbody(s) 2) Secondary Antibody Control(s).  OR – Consider multiple sections to be duplicate samples of the same antibody!
• Use enough antibody solution to cover the sample with a slight bubble.  Too much volume can overcome the hydrophobic barrier.
• Handle DAPI with care – it has mutagenic properties.  DAPI wastes, including washes, should be collected separately as chemical wastes.
• Remove slides one at a time from final PBS rinse.  Wick away excess solution and allow any remaining to evaporate, 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.