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NEW PELCO BioWave® Pro+ Microwave Tissue Processor
go to the Immunolabeling Application Kit

Poster From Life Technologies
Diane R. Gray, Julia F. Kottmeier, Mark A. Sanders, Laurel R. Stone, Matthew P. Beaudet
Life Technologies • 5791 Van Allen Way • Carlsbad, California 92008 • USA; Imaging Center • University of Minnesota, Twin Cities Campus • St. Paul, Minnesota 55108 • USA
Presented April 2009 in Denver, Colorado at the American Association for Cancer Research

Controlled Variables in Microwave Processing Leads to Improved Fluorescence Visualization Techniques

 

Fluorescent and DAB Labeling - Strategy for Confocal Applications

The protocol outlined below is based on:

Munoz, T.E., Giberson, R.T., Demaree, R., Day J.R. (2004) Microwave-assisted immunostaining: a new approach yields fast and consistent results. J. Neurosci. Methods, 137:133-139.

Ferris, A.M., Giberson, R.T., Sanders, M.A., Day, J.R. (2009) Advanced Laboratory techniques for sample processing and immunolabeling using microwave radiation. J. Neurosci. Methods 182(2):157-164.

DAB labeling Microwave Settings Fluorescent labeling
Step Time Wattage Step
1. Antigen retrieval 5 min 550

1. Antigen retrieval*
2. Buffer rinses 2 x 1 min 150 2. Buffer rinses
3. H2O2 3 min 150  
4. Buffer rinse 1 x 1 min 150  
5. Blocking step 3 mina 150 3. Blocking step
6. Primary antibody 8 minb 150 4. Primary antibody
7. Buffer rinses 3 x 1 min 150 5. Buffer rinses
8. Secondary antibody 8 minb 150 6. Secondary antibody
9. Buffer rinses 2 x 1 min 150 7. Buffer rinses
10. ABC incubation 6 minc 150  
11. Buffer rinses 2 x 1 min 150  
12. Imidazole rinses 2 x 1 min 150  
13. Color reaction As required

Bench

  
14. Imidazole rinses 2 x 1 min 150  
15. Buffer rinses 2 x 1 min 150  
a 3 min = 1 on - 1 off - 1 on.
b 8 min = 3 on - 2 off - 3 on.
c 6 min = 2 on - 2 off - 2 on.
*The antigen retrieval protocol we have found valuable is cited in both the Munoz et al. and Ferris et al. papers. The use of imidazole buffer rinses prior to and after the color reaction in the DAB labeling protocol is a beneficial addition.

Labeling Strategy for Confocal Microscopy

The fluorescent labeling protocol is used for Confocal labeling. The only changes to the protocol would be to accommodate an increase of tissue thickness. The protocol above has times that were used to label 50µm thick Vibratome sections of rat brain. The addition of vacuum during the labeling steps as well as an increase in magnetron on/off time are the adjustments to be made. For every minute of On time add 1 minute of Off time. The addition of Triton® X-100 to antibodies and buffer rinses will help the penetration of the antibody into the tissue. For more difficult to penetrate sample the antibody labeling cycle in the microwave can be repeated as necessary. Use continuous vacuum (20" Hg) for antibody incubations. The following link will provide pre-embedding labeling protocols for electron microscopy:

http://meshulspecial.files.wordpress.com/2010/07/imidazole-pre-embed_immuno_procedure.pdf

Typical labeling setup on the PELCO ColdSpot® Pro

Sequenza™ Slide Rack (#36105) and Coverplate™ (#36107) system for slide staining in the microwave.

A. A front view of the coverplate that is placed on the glass slide to form a capillary gap for the staining reagents to flow through. The arrow indicates the notch in the front clip of the coverplate that connects with the tab in Fig. 1C (arrow).

B. The rear view of the coverplate showing the tabs (<) that position and hold the slide (tissue side down) in position prior to placing the complex into the cassette base.

C. The cassette base holds the coverplate and slide complex together to maintain the capillary gap during microwave processing. The lid is not needed for microwave protocols.

Antibody steps require about 100µl of reagent with the system.

PELCO® Technical Notes, Coverplate™ Technology (128KB PDF)

12 or 24-well plates work best for free-floating sections. Only use enough reagent to just cover the tissue. 24-well inserts (36172) can be used to aid specimen transfer between antibodies and rinses.

Wellplate well inserts
Prod. #36172


Sequenza™ Slide Rack
on PELCO ColdSpot® Pro
PELCO Prep-eze™ Wellplate
on PELCO ColdSpot® Pro

When starting with paraffin sections the following microwave protocol can be used to deparaffinized the slides. 
Steps 1-3 are done on the ColdSpot similar to the basic microwave setup above.


Step Container -Step

Microwave Settings

Wattage/ Temp. Restrictions

 Time
1. Coplin Jar - 50ml xylene - deparaffinization 250W / None 4 min.
2. Coplin Jar - 50ml 95% ethanol - rehydrate 150W / 40°C 1 min.
3. Coplin Jar - 50ml 95% tap water- rehydrate 150W / 40°C 1 min.

 


Figure 6: Confocal projection of Elodea canadensis labeled with Hoechst 33258 nucleic acid probe (blue stain) for 6 minutes in the absence (A) and presence (B) of 150W microwave radiation. Confocal projection of Arabidopsis thaliana root tip labeled with Hoechst 33258 nucleic acid probe after 6 minutes of 150W microwave radiation (C). Confocal projection of in situ hybridization patters of whole chromosome probes (red) hybridized to nuclei of paraffin embedded rabbit skin (D). Confocal projection of mouse kidney paraffin sections labeled with anti-Factor VIII monoclonal antibody for 60 minutes on the bench (E) and after 6 minutes of 150W radiation (F) (Scale for all bars = 50µm) (Table 1). (Mark Sanders, Imaging Center, College of Biological Sciences, Univ. of Minnesota, St. Paul, MN). Reprinted with permission of Galvez et al., Microscopy and Analysis, Nov. 2006.





Figure 7. Retinas were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) overnight at 4°C. Following fixation the tissue was rinsed 6 x 20 minutes in buffer prior to beginning antibody labeling. The bench staining protocol required 7 days. The labeling results were completed in an afternoon using microwave-enhanced labeling during a workshop held at the Univ. of Minnesota Imaging Center (Mark Sanders, Director - May 17-19, 2006). The retinas were triple-labeled for:
  • Collagen Type IV (basal lamina surrounding blood vessels) with rabbit anti-type IV collagen and the secondary conjugated to FITC (green label)
  • Glutamine Synthetase (enzyme found in retinal Müller glial cells) with mouse anti- glutamine synthetase and the secondary conjugated to Cy3 (red label)
  • Glial Fibrillary acidic protein (GFAP an intermediate filament protein of astroglial cells) with chicken anti-glial fibrillary acidic protein and the secondary conjugated to Cy5 (blue label)
Primary antibody labeling was done at 170W for 12 minutes (4 on - 4 off - 4 on) under vacuum (15" Hg). Secondary antibody labeling was done at 170W for 6 minutes (2 on - 2 off - 2 on) under vacuum (15" Hg). Images were collected on a Nikon C1si Confocal Microscope.


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