Immunofluorescence - Protocols - Microscopy

Immunofluorescence Microscopy Using Frozen Sections, Chamber-slides or Coverslips

Frozen sections, 4 or 8-well chamber slides or cover-slips containing cultured cells will be fixed in 4 % paraformaldehyde in PBS for 30 min at room temperature (chamber-slides or cover-slips) in PBS containing 0.05%-0.1% Triton X-100. For examination of cell surface components, methanol or Triton X-100 extraction should not be used. After a 30-min incubation in PBS containing 0.05% Twen-20 (PBST) and 3%-BSA, samples are incubated with the affinity purified primary antibodies (~2 g/ml in PBST-BSA) for 1-2 hr at room temp. Samples will be washed in PBST 3 times and incubated with Cy2, Cy3 or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Inc.) for 1 hr. For control samples, no primary antibodies or pre-immune serum can be used under the same conditions. The samples should be well washed with PBS, mounted, and examined with a BioRad MRC 1024ES confocal laser scanning microscope.

TISSUE PREPARATION
For Immunofluorescence For Frozen Sections

  1. Take slide sections that were stored in -20°C and place slides into ice cold ETOH or MEOH for 5 min
  2. Air dry slides
  3. Rehydrate slide sections in PBS or TBS for 5min 2X
  4. Wash slide sections once in PBS-T or TBS-T (Tween 20 at 0.05%) for 5 min
  5. Block in 3% BSA in PBS-T or TBS-T for 1HR
  6. Rinse once in PBS-T or TBS-T for 5min
  7. Add Primary Antibody or Antibodies at your predetermined dilution in PBS-T or TBS-T + 1% BSA for 1-2 HR in a humidified chamber and cover with parafilm
  8. Wash in PBS-T or TBS-T for 15 min 3X
  9. Add your Secondary Antibody or Antibodies at your predetermined dilution in PBS-T or TBS-T + 1%BSA for no more than 1.5 HR in the dark in humidified chamber and cover with parafilm (For Unknown use CY2 anti Host of primary)
  10. Wash in PBS-T or TBS-T for 15min 3X

***IF using nuclear stain dilute in PBS-T or TBS-T for stain for 5 min
DAPI (1:1000)
Sytox Green (1:20,000)
Sytox 60
PI (1:1000 of stock)

  1. Wash once in PBS or TBS for 5 min
  2. Add 1 or 2 drops or Sigma Gel mount on center of slide and cover with cover slip and allow to dry on slide drying rack for 1 Hr at room temp in the dark
  3. Store slides at 4°C in a slide holder until analysis


For Parafin Sections

  1. Fix Tissue in 4% Paraformaldehyde (Fresh Made in PBS) for 1H at RT and 4°C overnight
  2. Wash in PBS for 30 min 3X
  3. Dehydration in 50% /ETOH, 70%ETOH (can store up to 1 week at 4°C)
  4. Send Sample for Parafin embedding in Diagnostic Center
  5. Take paraffin section in slide and put into Xylene twice for 5 minutes
  6. Put slide into 100% ETOH twice for 5 minutes
  7. Air dry slide


For Immunofluorescence For Parafin Sections

  1. Take slide sections that were air dried (#7) and wash twice in PBS or TBS for 5 min
  2. Wash slide sections once in PBS-T or TBS-T (Tween 20 at 0.05%) for 5 min
  3. Block in 3% BSA in PBS-T or TBS-T for 1HR
  4. Rinse once in PBS-T or TBS-T for 5min
  5. Add Primary Antibody or Antibodies at your predetermined dilution in PBS-T or TBS-T + 1% BSA for 1-2 HR in a humidified chamber and cover with parafilm
  6. Wash in PBS-T or TBS-T for 15 min 3X
  7. Add your Secondary Antibody or Antibodies at your predetermined dilution in PBS-T or TBS-T + 1%BSA for no more than 1.5 HR in the dark in humidified chamber and cover with parafilm (For Unknown use CY2 anti Host of primary)
  8. Wash in PBS-T or TBS-T for 15min 3X

***IF using nuclear stain dilute in PBS-T or TBS-T for stain for 5 min
DAPI (1:1000)
Sytox Green (1:20,000)
Sytox 60
PI (1:1000 of stock)

  1. Wash once in PBS or TBS for 5 min
  2. Add 1 or 2 drops or Sigma Gel mount on center of slide and cover with cover slip and allow to dry on slide drying rack for 1 Hr at room temp in the dark
  3. Store slides at 4°C in a slide holder until analysis