High Resolution Fluorescence Microscopy

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Frequently Asked Questions

How to contact us?

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DNA Sequencing & Synthesis: 
Email Core Lab  
or phone: 503-494-2472.

Microscopy: 
Email Aurelie Snyder  
or phone: 503-494-6994.

For technical assistance and admin: 
Email Tom Keller, PhD  
or phone: 503-494-2442.

Did you know?

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We can help troubleshoot your results from all Core Facility services: DNA sequencing, synthesis, and microscopy. If we make a mistake we will correct it free of charge.

Resource Links, Notes & Protocols Menu

Microscopy Methods  - Coverslips  - Mounting Media  - Light Sources  - Standard Filter Set  - Live cell filter set  - Quantum Dots Buffers and Treatments to Reduce Autofluorescence  - 20x MOPS  - Sodium Borohydride Block treatment  - Sudan Black treatment  - Trypan treatment Cell Biology  - Cell Cycle Synchronization  - Sodium Phosphate Buffer  - Chondroitinase ABC Buffer  - 4% Paraformaldehyde Immuno-Fluorescence  - Fix and Stain cells grown in culture  - Fix and Stain Vibrotome Sections  - Paraffin, Cryo or Thin Vibrotome Sections

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Microscopy Methods

We specialize in live-cell, real-time fluorescence microscopy using the Applied Precision DeltaVision™, image restoration system and a Bio-Rad confocal.

Coverslips

The proper coverslip to use is a #1.5. All objectives have been corrected to this thickness. Many lab stores carry a #1 which is incorrect: although it does not make your specimen noticeably different to your eye, it does affect the resolution of the images.

Mounting Media

Several mounting media are not compatible with certain dyes and/or fluorescent proteins. Always read the product info before using. Currently, in the Core Lab, we use Fluoromount-G (refractive index 1.4) from Southern Biotech which we get through Fisher Scientific Co..
Examples:
Phenyladiamine is not usable for Cyanine dyes. This is the main ingredient in Vectashield brand mount.
Crystal Mount is not good for several red dyes (PE, PC, APC) or fluorescent proteins.
Prolong is good for Alexa dyes but may be bad for fluorescent proteins. We suggest testing before wholesale use.

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Light Sources

The Xenon Arc Lamp

The Mercury Arc Lamp

These show the emission spectra of a mercury arc lamp and a xenon arc lamp.
Note differences in peaks and intensities for any given wavelength. This is why Dapi (360) FITC (488) and Rohodamine (568) have been the colors of choice. Xenon seems to have the answers as it has a more even emission but in general they are also lower in intensity so you may not be able to see your dye.

Standard Filter Set

Description of the Standard Filter Set for the Applied Precision CoreDV

The standard filter set for the MMI COREdv microscope allows 4 colors from UV to far red. Excitation is represented by dotted lines and emission is shown by solid lines. The polychroic is shown in black.
Each filter designation is the peak wavelength in nanometers followed by the width which is equally divided on either side of the peak. (e.g., Dapi excitation happens from 340-380nm)

DeltaVision CoreDV Standard Filter Set

Excitation peak and width	Emission peak and width
360 +/- 20 nm	457 +/- 25 nm
490 +/- 10 nm	528 +/- 19 nm
555 +/- 14 nm	617 +/- 37 nm
640 +/- 10 nm	685 +/- 20 nm

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Live cell filter set

Description of the Live Cell Filter Sets for the Applied Precision CoreDV

The Live filter sets for the MMI COREdv microscope allow 2 colors each in pairs optimized for separation. Excitation is represented by dotted lines and emission is shown by solid lines. The polychroic is shown in black.
Each filter designation is the peak wavelength in nanometers followed by the width which is equally divided on either side of the peak. (e.g., Cyan excitation happens from 418-442nm)

Live Cell Filter Set for the Applied Precision CoreDV

Fluorescent proteins
Cyf and Yfp Excitation peak and width Emission peak and width 430 +/- 12 nm 470 +/- 12 nm 500 +/- 10 nm 535 +/- 15 nm
Gfp and mCherry Excitation peak and width Emission peak and width 470 +/- 20 nm 525 +/- 25 nm 572 +/- 18 nm 632 +/- 30 nm

It is not recommended to combine more colors as the excitation and emmission spectra of the neighboring colors overlap enough to allow bleedthrough.

Colors shorter than Cyan are not recommended due to the damage to living cells caused by excitations less than 400nm.

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Quantum Dots

These nanoparticles are very bright and not subject to photobleaching. They may be the answer to detection problems where epitope numbers available for normal antibody tagging are limited. However, there are caveats. First, you must have the correct dichroic mirror or laser to excite them. Secondly, you should not mix dots with other types of fluorescent labels; mixing will result in many headaches. For instance, when you have a red or far-red dot combined with FITC or a GFP you will photobleach the green because the excitation for the dots is lower than the FITC or GFP and the dichroic allows all that light through. Also, if you had to switch dichroics and you have a system with separate filter wheels, this can create a problem with pixel registration. Invitrogen and Evident sell Quantum dots.

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Buffers and Treatments to Reduce Autofluorescence

20x MOPS

41.9 g MOPS (Morpholinopropanesulfonic acid (free acid))
6.8 g sodium acetate (MW 136.08)
2.6 g EDTA (MW 372.24)
400 ml DEPC H2O
Adjust pH to 7.2 with NaOH and QS to 500 ml with DEPC-treated H 2O. Use at 1x. (A.S., 10/2005)

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.

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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

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Cell Biology

Cell Cycle Synchronization

Use excess thymidine 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

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

Key point: Make fresh each time needed.

4% Paraformaldehyde

100 ml PBS 50mM pH 7.4
4 gm paraformaldehyde
Heat in the hood on a magnetic hotplate stirrer on about medium but never let the solution get over 59C. Higher than this will degrade the PFA.
It will take 30 to 50 minutes. If there are a few persistent crystals, dissolve them with a few drops of 1 N NaOH.
Cool the solution to room temp and adjust pH back to 7.4 with a few drops of 1 N HCl.
Store at 4o C and use within one month.
Some labs have acceptable results by allocating it to 15 ml tubes and freezing it at -20C for up to 1 year.
Sterile filtering can prolong its life as well.

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Immuno-Fluorescence

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!

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!

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

The most convenient way to schedule microscopy work is email the microscopist.

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