Histology and immunofluorescence staining and analysis

Immunofluorescence Staining – Paraffin Sections

Protocol by: Derek Sung (adapted for Kahn Lab, updated 2/5/2024)

Deparaffinization & Rehydration

1. Place slides in xylene for 45 minutes minimum (or 10 minutes 3x)
2. Place in 100% EtOH for 15 minutes
   1. Optional: briefly dunk in a separate container of 100% EtOH to wash off excess xylene
3. Place in 95% EtOH for 10 minutes
4. Place in 70% EtOH for 10 minutes
5. Place in DI H₂O for 5 minutes

Antigen Retrieval & Blocking

1. Place slides in sodium citrate antigen retrieval buffer and microwave as follow: [1] 3.5 minutes on high (or until boiling) & rest for 3 minutes, [2] 1.25-1.5 minutes on high and rest for 3 minutes and repeat 4x (5 times total)
   1. Note: these times are for the Kahn Lab microwave using the glass slide holder in a P1000 box filled up to the line with Citrate Buffer. Times may vary depending on strength of microwave, check in between cycles to make sure slides are still covered in buffer. The buffer should be boiling in the last 5-10 seconds of each cycle (any longer it will likely boil over)
   2. Sigma Citrate Buffer Antigen Retriever 10x C9999
2. Let cool for at least 45 minutes (should be around room temperature or just slightly warm)
3. Pour off antigen retrieval buffer (do not reuse)
   1. Optional: wash briefly with 1X PBS
4. Dry slides with kimwipe, careful not to wipe over the section but do not let sections try
5. Outline sections with hydrophobic marker and allow ink to dry (briefly)
6. Apply blocking solution: 10% normal serum (goat or donkey) in 1% BSA (prepared beforehand)
   1. Note: if you are using goat antibodies (primary antibody raised in goat), you CANNOT use goat serum or you will get non-specific staining
7. Incubate for 1 hour

Primary Antibody

1. Prepare primary antibody solution at correct concentrations & species in 1X PBS
2. Remove blocking solution and drain excess with kimwipe (again, careful not to touch the section)
3. Apply primary antibody immediately and place in 4°C fridge covered, overnight
   1. Note: some protocols say 2 hours at room temperature or in an oven works, this typically results in weaker signals so would not recommend it

Secondary Antibody

1. Remove primary antibody and wash in 1X PBS as follows: one quick wash + two 5-10 minute washes on a rotator
2. Prepare secondary antibody solution at correct concentrations & species + DAPI in 1X PBS
3. Dry slides with kimwipe, careful not to wipe over the section but to not let sections try
4. Apply secondary antibody and incubate at room temperature for 30 minutes (but no longer than 1 hour)

Mounting

1. Remove secondary antibody and wash in 1X PBS as follows: one quick wash + two 5 minute washes on a rotator
2. Dry slides with kimwipe, careful not to wipe over the section but do not let sections try
3. Add antifade reagent (typically ProLong Gold or Diamond), 1-2 drops per section
   1. Try not to get any bubbles, carefully remove with a pipet if there are
4. Let glass coverslip drop on top of section, let the reagent spread, leave to dry overnight
   1. For same day use: apply some nail polish (not too much) on top and bottom to stabilize the coverslip, let it dry for at least 30 minutes
5. Slides are now ready for imaging