Welcome! I hope you’ve completed the previous article on using a light microscope. If not, I recommend that you read it. Today, I will discuss permanent slide preparation for microscopic study.
This is the most common procedure used in the study of veterinary histology. You do not need to memorize the entire permanent slide preparation process. Instead, focus on understanding the basic steps of permanent slide preparation. Let’s begin with the basics.
I won’t go into detail about permanent slide preparation here. For more details, please follow your class lectures or read books.
Learning requirements
You should know these topics before starting permanent slide preparation –
- Tissue and collection procedure of tissue from the animal body
- Different types of fixatives and their general properties
- Details on other chemical agents like alcohol, xylene, and paraffin
- Function of the microtome machine, water bath
- Different types of staining dyes and their staining properties (acidic and basic dyes)
- Details on mounting agents and finally
- Different parts of the microscope with their uses
Okay, now you may start.
Permanent slide preparation (steps of tissue preparation)
You should know what happens at each step of permanent slide preparation for microscopic study. You can find more information in books and online about permanent slide preparation and the steps of tissue preparation. Here, I will simplify the procedure. Let’s start.
This article is for beginners who want to know the basics of permanent slide preparation.
Suppose you have tissue and need to study it under a microscope. The process has several key steps. First, slice the tissue into thin sections and attach them to a glass slide. This involves specific procedures, which we will discuss next.
Since most tissue samples are colorless, the second step is to color these attached sections with specific dyes. This is called staining. Finally, to preserve the tissue on the slide for a long time, place a cover slip over it using a mounting agent. I hope you now see the basic steps of permanent slide preparation. To summarize, this process has three main steps:
Tissue preparation (include tissue collection to attachment of this tissue on a glass slide)
Staining of those tissues attached to a glass slide (as most of the tissues are colorless), commonly done using acidic or basic dye.
Mounting (using a cover slip and mounting agent; for the protection of stained tissue on a glass slide)
Now that you know the three main steps, let’s walk through what you should do at each stage, starting with tissue preparation.
Tissue preparation
In this step, your goal is to collect tissue from the animal, cut it into thin pieces, and attach it to a glass slide. Do the following:
First, collect a small piece of tissue, about 10 by 5 millimeters. Use a biopsy or surgery to collect it. If you keep the tissue too long without chemicals, it will break down from enzymes or bacteria. To prevent this, use agents like formalin, acetone, or acetic acid.
These are fixatives, and the process is fixation. Fixatives preserve tissue structure and shape. In our lab, we often use 10% buffered formalin and Bouin’s solution. After using fixatives, rinse away any excess with running tap water.
“You should know the composition of 10% buffer formalin and Bouin’s solution with their functions.”
Now your tissue contains water. You need to embed it in a solid medium. Usually, liquid paraffin is used. Use the microtome to slice the tissue for the next steps. Paraffin does not mix with water, so first remove water by dehydrating with alcohol. After dehydration, make the tissue see-through using xylene. This step is called clearing. Alcohol is replaced by xylene here.
Infiltrate the tissue with melted paraffin at 58–60°C. Swap the clearing agent xylene for paraffin; this is infiltration. Use an embedding box or two L-shaped molds. Fill them with melted paraffin. Place the cleared tissue into the melted paraffin in the box. Refill the box or molds. This is called embedding the tissue.
Now the embedded tissues are ready for thin sectioning. Use a hand microtome for this process, called sectioning. Float the tissue sections in a water bath. Stick the thin tissue section to a glass slide with glue. Dry the slide with the tissue at 37°C in a slide dryer.
So, we may conclude the steps of tissue preparation into the following –
- Collection of tissue
- Fixation of tissue
- Dehydration of tissue
- Clearing of tissue
- Infiltration of tissue with paraffin
- Embedding of tissue in paraffin
- Sectioning of tissue into thin slices
- Floating of section into the water bath
- Attachment of thin tissue on a glass slide
After completing the tissue preparation steps, let’s move to the next process: staining the colorless tissue.
Staining of tissue
Follow the normal method for staining tissue. Use basic or acidic dyes. Methylene blue is a basic dye, and eosin is an acidic dye; these are common choices.
If we use Hematoxylin and eosin for staining, use the normal lab procedure. Please look for the permanent slide preparation PDF (staining).
Here, we need to perform the following –
- After embedding with paraffin, remove the paraffin to stain the tissue.
- Again, we need to rehydrate the tissue section.
- We should use running tap water for washing.
- Now use the hematoxylin stain.
- Again, wash the tissue with running water.
- Now use the eosin stain and wash with running water.
- Now rehydrate the tissue again.
- Finally, clearing with xylene.
With staining complete, we’ll now proceed to the final step: mounting the slide.
Mounting of the slide
Dry the stained slides. Protect the tissue by putting a cover slip on it with a mounting agent. In our lab, we often use DPX, made from dextrin, plasticizer, and xylene.
Conclusion
You should now have a basic idea of how to make a permanent slide for microscopic study. Now, you can try preparing a permanent slide by following each step in order.
