For us in histology, normal tissue identification is typically an easy process. As histologists, we were once required to identify all normal tissues by H&E under the microscope.
For abnormal tissue however, an H&E stained slide can be very difficult to identify tissue type and diagnosis. That’s why when a pathologist orders multiple stains on one tissue, they are trying to get a larger picture to understand its etiology.
To help in understanding, here are 3 stains on the same tissue, H&E, SMA, & CD8 at 4x & 10x. This is human tissue. Can you identify this tissue and its diagnosis?
We do a fair amount of special and fluorescent staining that requires aqueous mounting media and not the toluene based glue. Normally for special stains I make up a glycerin jelly that has phenol and must be kept in the refrigerator. This was taught to be the standard of aqueous cover slipping media. It never dries, sticks to everything and smells terrible.
The fluorescent slides need something different, usually a special fluor mount bought at one of the large chemical companies. This mount allows for very little light distortion while viewing through a con-focal or fluorescent scope. The amount of light distortion is called refractive index. Some of these mounting medias that having a low refractive index of 1.47 can be very expensive, $162.00 plus shipping for 10 ml.
10 ml may be enough to cover slip 300 slides if using only 30 um per slide but that adds $.60 to the cost of every slide. That may not seem like much but when it’s added to the total after processing, embedding, cutting, antibody plus working hours it can push the average researchers budget to the edge.
I thought, there has to be something better than this. Large companies get most of their protocols from the old histology books and change 1 micro-gram and it becomes proprietary. Then the price depends on demand and repeat customers (way too high). This is what I call commercial brutality.
I’m way to enterprising to pay these prices and be happy about it. Therefore I too will develop a protocol from the books and change one aspect of it and call it my own. The difference is, I will formulate it, test it, have others test it, then make it available to all the labs I have daily contact with (10) and help them save money.
One way to stop this money merry go round is to limit profits from unbelievable pricing on medical products. Paying $500.00 for a water bath that lasts 10 years is absurd! $162.00 for 10 ml of mounting media is too much!
I have made 25 ml the histo-lo-gis-tics version of aqueous mounting media for about $20.00, but could be 100 ml for $40.00. That’s the cost if you make it for yourself. It’s a room temp, permanent media that will last for 2-3 years with anti fading agent and refractive index of 1.46. So far it’s been tested by 2 independent labs in fluorescent microscopy and passed with no distortion. 2 more labs will be testing it this week and mine soon.
After testing is done, I will update the results, good or not good and post the recipe if it’s useful.
Background: The tissue and control are on the same slide, and the control is staying on but tissue is falling off.
There’s a lot of could be answers, that you might have to try before finding the answer. So here is a list, answers by histologists around the world.
Try not baking the control tissue just let them air dry, the humidity in the slide drying oven might be messing with the super frost plus slides. It’s just a coating on the slide of some sort, after the first dip they don’t work again like the first time, so just air dry the controls and bake after patient tissue is picked up.
Water is always a potential issue. Tissue can be finicky though. Have you tried poly-L-lysine coated slides? They do help is some occasions.
If you are drying your slides in the oven, check the temp and
Try going longer and tap off excess water before drying.
If the sections are coming off the slides entirely, or does it have a “chewed up” appearance? If the sections are coming completely off, I would agree with the others that water is the issue.
Also, make sure you are cutting no thicker than 4 microns, and let the sections really flatten out on the water bath for a minute.
Bake them well.
If the sections have more of a chewed up appearance, then it’s probably a combination of factors: fatty tissue, not well fixed, strong antigen retrieval.
After baking, “post fix” some of the IHC slides in formalin for about 15 minutes. Rinse in tap water then put on stainer as usual. This seems to help when certain antibodies like to chew up the tissue.
Another thing to try is different slides. There issues with bad batches/lots of slides – even the Fisher Superfrost plus.
Make sure the PM is done on the machine quarterly.
Our slides are the most important pieces of the project. Great care is always taken when shipping all slides using a small cell rectangular cut bubble wrap inside the box on top of the slides, then wrap the entire box in bubble wrap a few times around. This gives the safest way possible to eliminate broken slides. It’s the best skill to teach your interns, cutting bubble wrap, boxing it up and bringing to fed ex, then picking up coffee on the way back! Some slide boxes come with bubble wrap from the vendor, we save those and use when shipping also.