Here are recipes for fixatives that work well
FIXATIVES for Light microscopy
Davidson’s Fixative
70% Ethanol (66%) 100 ml
37-39% Formalin (22%) 33 ml
Glacial Acetic Acid (12%) 18 ml
Fekete’s Fixative
70% Ethanol (87%) 100 ml
37-40% Formalin (8.7%) 10 ml
Glacial Acetic Acid (4.5%) 5 ml
EM FIXATIVE
2% GTA/ 3% PFA IN 0.1M PHOSPHATE BUFFER (Grier)
50% EM grade glutaraldehyde 20 ml
20% Paraformaldehyde 75 ml
Distilled water 155 ml
0.2M Phosphate buffer, pH 7.4 250 ml
TOTAL VOLUME 500 ml
Hi Maria
Are you asking how best to stop the retina detaching during histology, or how best to fix the eye? As the previous answers have dealt with fixing, I will try to answer how best to stop the retina detaching.
During histological processing (paraffin, wax or frozen), you may notice that the retina has detached or moved away from the RPE / choroid. Usually this is due to too much force being used when killing the animal, perfusing the animal or extracting the eye. If you sacrificing the animal before fixation, try using a different method to cervical dislocation; this can often lead to retinal detachment. If you are perfusing the animal, decrease your flow rate if using a pump, or manually decrease the pressure you perfuse with (if using a needle and syringe). We typically perfuse with ice-cold saline, followed by ice-cold 4% PFA slowly and have no issues with the retina detaching. If you are removing the eye using forceps, try cutting the eye underneath the socket first to avoid torsion on the eye.
Hope this helps
Pete
The eye requires special processing because of the delicate nature of some of its parts and the toughness of others. It must be thoroughly fixed. Add 4% phenol to the lower percentage alcohols in order to soften the sclera and lens. Use chloroform as a clearing agent instead of xylene because it causes minimal shrinkage thus keeping the retina attached.
It depends very much on the species. 'Lower' vertebrates, such as fish, amphibia and reptiles, but others also, undergo retinomotor movements. Thus, a dark adapted retina easily falls away from the RPE. A retina exposed to light for an hour (especially during daytime) will remain firmly attached.
I work with ocular pathologies in MA and have obtained good results in fixation with Davidson. Besides the ocular globe become rigid, by facilitating the cut in the microtome.
Place the intact globe in Davidson for 24 hours, after making the cut, and maintained for 12 hours. Transferring to 70% alcohol.
Glacial acetic acid: 100 ml
Ethyl Alcohol 95% 300mL
Neutral buffered formalin 10%: 200 ml
Distilled water: 300 ml
It depends whether you are proceeding with cryosectioning or paraffin sectioning because fixation for both the methods vary.
For cryosectioning you dont need to perfuse the animal but it is important to remove the lens before fixing it in 4% PFA followed by sucrose solution. The embedding solution such as NEG 50 is excellent choice. I personally never had any problem of retina detachment with this method.
For paraffin sectioning it is important to perfuse the animal before removing the eye with ice cold 4% PFA. This way retina will remain firmly attached.
Retinal detachment during processing is a common problem, especially if you prepare vitrectromized eyes. Davidson`s fixative improves the situation. Unfortunately Davidson's is not compatible with immunohistochemical staining. Formalin fixation results in very successful results if you carefully enucleate the eye (using a surgical microscope), then place an incision few millimeters posterior to the corneal limbus. First gently inject formalin through the incision into the globe. Then inject fluorinated decalin throught the incision until the intraocualr fluid is completely exchanged by decaline. Leave the eye for several hours in formaline. Then the retina will remain attached.
I am having problems with human eye to prevent retinal detachment during processing for paraffin. I am doing IHC so unfortunately Davidson's would not work. Any suggestions?
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