Dr. Heyers:

Sorry it took so long to answer your post. But hopefully the following information will be helpful.

EGFP in paraffin: Direct visualization of fluorescence
1) Fixation: We use 10 % neutral buffered formalin at room temperature (25 - 27 oC). Recall that adequate fixation requires control of tissue thickness, fixative volume and time in fixative.

2) Dehydration in graded alcohols to absolute ethanol: We process "by hand" using one immersion in 70 % ethanol (for soft tissues) followed by two changes of 95 % ethanol followed by three changes of 100 % ethanol. We test for complete dehydration and lipid removal by testing 10 ml of the last "spent" ethanol in 90 ml of deionized water in a 100 ml graduate. If the ethanol does not remain clear upon striking the water, we continue the 100 % ethanol changes until the test is clear. Time in each alcohol step varies with tissue thickness and type, e.g. soft, small tissues 30 - 45 minutes each change; large, decalcified bone 1 - 2 hours & occasionally overnight in the last 100 % ethanol.

3) Tissue clearing with hydrocarbon solvents and paraffin infiltration: We use three changes of absolute methyl salicylate routinely for clearing our tissues. We find that it works well for decalcified bone. We have found no deleterious effects when used for processing soft tissues, e.g. uterus, heart, liver, brain, etc. The trick is the complete removal of the methyl salicylate in the first infiltrating paraffin. The advantage to using methyl salicylate is that its odor is distinctive and monitoring the second infiltrating paraffin for salicylate odor is easy to do. When an odor is noted in the second paraffin, discard the first paraffin and rotate the remaining paraffins down, e.g. 2 becomes 1, 3 becomes 2 and a new pot is melted for the third change. As with the above steps, time in the clearing and paraffins is dependent on tissue composition and size. Small soft tissues may spend 30 - 90 minutes in each salicylate change and 30 minutes in paraffin 1, 45 minutes in paraffin 2 and 60 - 90 minutes in para

4) Direct visualization of EGFP fluorescence: The blocks are sectioned at 3 - 5 microns, floated on a warm water bath and captured onto silane coated slides. The section is drained vertically approximately 5 minutes then annealed to the slide on a two stage warming platform. The first stage is thermostatically controlled to the melt point of the embedding paraffin (in our case 55-57 oC) the second stage is controlled to 60 - 62 oC. The adherent section is allowed to come to room temperature on a flat surface before further processing. The slide is deparaffinized in three changes of absolute xylenes five minutes each and cover slipped using permanent mountant. If counterstaining is required, the deparaffinized slide is rehydrated to deionized water and DAPI - Vectashield (Vector Labs. Various fluorescent mountants are also available from Molecular Probes.) mountant is applied prior to cover slipping. We view fluorescence using a Zeiss Axiovert Confocal Microscope or our old Leitz fluorescence scope. Images a

Immunostaining for EGFP on paraffin sections:

Steps as above to deparaffinized slide in deionized water. Zymed mouse anti-EGFP (Clone C163) has worked well for us on deparaffinized sections. The recommended start point is 2 - 5 ug/mL. This is an un-conjugated antibody. Therefore you can choose secondary antibodies to enhance the detection of the primary interaction. We've had our best success using biotinylated anti-mouse IgG followed by Strepavidin-Texas Red for fluorescence or Strepavidin-horseradish peroxidase for light microscopy.

Hope this helps.
Kindest regards, 

Donna Montague, M.S. Research Associate
Center for Orthopaedic Research
University of Arkansas for Medical Sciences
Little Rock, AR, USA 

-----Original Message-----
From: oliver heyers [mailto:oliver_heyers@yahoo.com] 
Sent: Thursday, November 14, 2002 4:48 AM
To: MontagueDonnaC@uams.edu
Subject: GFP in paraffin sections


Hi Dr. Montague
I have found your name by searching the internet for
EGFP visualisation in paraffin embedded tissues. They
say you are a bit of an expert in this method.
There fore I am writing: We like to visualize GFP in
parasites in intermediate host snails- the expression
rate is very low, therefore we like to find
transfected cells using anti-GFP antibodies in
paraffin sections- with these sections histological
staining is easier and the visualisation should be
better than in thicker cryo sections. Do you have a
protocol for fixation of tissues and paraffin
embedding visualize GFP expression by using antibodies
?
Thank you very much for your help !
Best regard
Oliver Heyers

Humboldt University
Institute of Molecular Parasitology
Berlin
Germany

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