Protocols

• Autoflourescence
• Cell Biology
• Buffers
• Immunofluorescence
• Mounting Media

Sodium Borohydride Block treatment

N.B. Make Fresh always

1. Measure out 2 separate 10 mg aliquots of NaBH₄
2. Have ready two tubes with 10 mL of PBS or TBS, pH 8
3. Right after washing out fixative, dissolve one aliquot in the buffer and put it on specimen.
4. Let sit
   
   • 5 minutes for cells
   • 10 minutes for thin sections
   • 15 minutes for filters
   • 50 minutes for thicker vibrotome sections
5. Repeat with second aliquot of NaBH₄ in buffer
6. Continue with regular preparation for immunofluorescence microscopy

Key points: 1 mg/mL NaBH₄ in buffer, pH 8, prepared just before use.

Sudan Black treatment

1. Use 0.3% Sudan Black (w/v) in 70% ETOH (v/v) applied to slide for 10 minutes after the secondary antibody application.
2. Rinse quickly with PBS 8 times and mount
3. For FITC and Alexa 594 this does not reduce the emission signal noticeably.

References

• Doyle, K. et. al. (2003) Working With GFB In The Brain, BioTechniques 34:492-494, March
• Schnell, S. et. al. (1999) Reduction of Lipofuscin-like Autofluorescence in Fluorescently Labeled Tissue , J. Histochemistry& Cytochemistry Vol 47(6): 719-730

Trypan treatment

1. Trypan Blue 250ug/ml, pH 4.4
2. Dye solution is removed after 1 minute
3. Wash in buffer quiickly and mount in aqueous/glycerol mounting media.

Reference

Chok P. Wan, Choon S. Park and Benjamin H. S. Lau A rapid and simple microfluorometric phagocytosis assay, Journal of Immunological Methods Volume 162, Issue 1, Pages 1-7

Cell Biology

Cell Cycle Synchronization with Double Thymidine

Used to synchronize cells (Hela) at the G1/S transition.

1. Add 2 mM thymidine to the media, leave it on overnight.
2. Put the cells in thymidine-free media for 10-11 hrs the next morning.
3. Add 2 mM thymidine back in the evening.
4. The next day, harvest them in 2 hr intervals.

Reference

• Qiao, Fengyu, Moss, A. and Kupfer,G.M. Fanconi Anemia Proteins Localize to Chromatin and the Nuclear Matrix in a DNA Damage- and Cell Cycle-regulated Manner. Journal of Biological Chemistry Vol. 276, No.26, June 29, 23391- 23396, 2001

Sodium Phosphate Buffer, 100 mM, pH 7.4

1. Weigh out
   • 240 mg NaH₂PO₄ (monohydrate with FW = 120 g/mole)
   • 1.2 g Na₂HPO₄ (dihydrate with FW = 142 g/mole)
2. Dissolve in 100 mL double-distilled, deionized H₂O

Chondroitinase ABC buffer

1. Prepare 0.03M NaAc in Tris, pH 8.0
• 250 mg Sodium-Acetate in 100 mM Tris HCl pH 8.0
2. Use this to dilute Chondroitinase ABC to 0.1 U/mL
3. Warm to 37C

Fix and Stain cells grown in culture

1. Rinse cultures with 100 mM TB pH 7.6 buffer (henceforth "plain buffer")
2. Fix cells with 4% paraformaldehyde, pH 7.4 for 1 hour
3. Wash 3 x 5 minutes with plain buffer
4. Digest 15 minutes with 0.1 U/mL Chondoitinase ABC^a in ChABC buffer at 37C
5. Wash 3 x 5 minutes with 100 mM TB pH 7.6 buffer containing 0.1% Triton-X^b
6. Block in 2% normal serum of the same host as the 2nd antibody for 15 minutes
7. Incubate 1-2 hours at room temp. with primary antibody^c
8. Wash 3 x 5 minutes with plain buffer
9. Repeat block in 2% normal serum of the same host as the 2nd antibody for 10 minutes
10. Incubate with 2nd antibody + fluorophore, e.g., FITC, for 30 minutes
11. Wash 3 x 5 minutes in plain buffer
12. Counterstain (if desired) with DNA dye^d (Hoescht, DAPI, other) at 2 microgram/mL for 10 minutes
13. Wash 3 x 5 minutes in plain buffer
14. Equilibrate in Slow Fade buffer for 5 minutes
15. Coverslip with Slow Fade in glycerol or use some other anti-fade agent

Notes

• Total time ~5 hours
• a. May have to use a different digestion enzyme, or none depending on the primary antibody,
  e.g., heparitinase works better for laminin than Bovine testicular hyaluronidase (BTH)
• b. Tween-20 or NP-40 can be substituted for Triton-X
• c. If you are using more that one primary antibody, be sure they are not made in the same animal.
  You must have different host for each primary. You may have the same host for the secondary antibody
  as long as the labels are different so you can distinguish each primary :-)
• d. Some commercial anti-fade reagents come with DAPI DNA stain.
• Plan your controls ahead of time ;-) :-)
  
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Fix and Stain Vibrotome Sections

1. Rinse sections 3 x 10 minutes with 100 mM TB pH 7.6 buffer (henceforth "plain buffer")
2. Put sections in glass bottle
3. Wash 30 minutes in plain buffer + 50 mM EDTA at 37C
4. Digest 30 minutes with 1 ug/mL Protease-K^a in 100 mM Tris-HCl, pH 8 + 50 mM EDTA at 37C
   Alternative permeabilization (steps 3 and 4)
   • Incubate 30 minutes with ABC Tris-Acetate buffer, pH 8, at 37C
   • Digest 50 minutes with 0.1 U/mL Chondoitinase ABC^a in ChABC buffer at 37C
5. Incubate 20 minutes with 100 mM TB pH 7.6 buffer containing 2 ng/mL glycine
6. Return sections to 24-well plate
7. Wash 3 x 10 minutes in plain buffer
8. Block for 30 minutes in 2% normal serum of the same host as the 2nd antibody in plain buffer
9. Incubate 48 hours, with gentle agitation at 4C, with primary antibody^c
10. Wash 3 x 5 minutes with plain buffer
11. Repeat block in 2% normal serum of the same host as the 2nd antibody for 10 minutes
12. Incubate overnight, with gentle agitation at 4C, with 2nd antibody + fluorophore, e.g., FITC
13. Wash 3 x 10 minutes in plain buffer
14. Counterstain (if desired) with DNA dye^d (Hoescht, DAPI, other) at 2 microgram/mL for 20 minutes
15. Wash 3 x 10 minutes in plain buffer
16. Equilibrate in Slow Fade buffer for 5 minutes
17. Mount coverslip with Slow Fade in glycerol or use some other anti-fade agent
18. Seal with nail polish or alternative.

Notes

• a. May have to use a different digestion enzyme, or none depending on the primary antibody,
  e.g., heparitinase works better for laminin than Bovine testicular hyaluronidase (BTH)
• b. Tween-20 or NP-40 can be substituted for Triton-X
• c. If you are using more that one primary antibody, be sure they are not made in the same animal.
  You must have different host for each primary. You may have the same host for the secondary antibody
  as long as the labels are different so you can distinguish each primary :-)
• d. Some commercial anti-fade reagents come with DAPI DNA stain.
• Plan your controls ahead of time ;-) :-)
  
  Return to top.

General immuno-staining of paraffin, cryo, or thin vibrotome sections^*

1. Deparaffinize sections to buffer; or hydrate cryo sections to buffer; or put vibratome sections in buffer.
2. Wash 3 x 10 minutes in buffer.
3. Permeabilize with chosen enzyme. This must be optimized for the desired protein but is usually about 30 minutes.
   Protease-K, 0.5 - 1%, is the usual but your mileage may vary.
4. Wash 3 x 10 minutes in buffer.
5. Wash in buffer 2 x 10 minutes
6. Block in 2% serum from the secondary antibody host in buffer.
7. Primary antibody dilutions must be optimized for the particular antibody but a good place to start is 1:100.
   Apply antibody to tissues in a moist chamber for 24 or 48 hours at 4C on a shaker table (gentle agitation).
8. Wash 3 x 10 minutes in buffer.
9. Block again in 2% serum.
10. Apply secondary antibody with label for 6 hours to 24 hours at 4C, with gentle agitation.
    Dilution is proportional to 1st antibody (start at 10x but need to be more dilute).
11. Wash 3 x 10 minutes in buffer.
12. Wash 3 x 10 minutes in buffer.
13. Mount in antifade agent (for fluorescence).

Notes

• Buffers can be 50-100 mM phosphate buffered saline pH 7.4, 100 mM Tris-HCl pH 7.6 or 100mM Tris-Maleate pH 7.6.
• Permeabilization may be done with Protease K, Chondroitinase ABC, Pepsin, Hyaluronidase, etc.,
  but you MUST optimize for tissue and protein.
• Adapted from Antibodies: A Laboratory Manual by Ed Harlow and David Lane; 1988, Cold Spring Harbor Laboratory Publications
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General Comments
Several mounting medias are NOT compatible with certain dyes or fluorescent proteins. Always read the product info before using.

Keep in touch

Please email the journal citation to Tom Keller at kellert@ohsu.edu For work supported with confocal images from the OHRC CLSM, please also email citations to Peter Steyger at steygerp@ohsu.edu

Thanks.

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References and Links

• Protocols Protocols for immuno-fluorescenc microscopy.
• NIH-Image homepage Information on the (free) NIH-Image analysis program.
• Excitation & Emission Maxima Fluorescent Dye emission Chart (courtesy of Lance Ladic, UBC.
• Principles of Confocal Microscopy from the University of Waterloo
• API Deltavision Homepage