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How To Fix Isolated And In Situ Primary Cells

Posted in: Microscopy and Imaging

Unlike immortalized cells lines, primary cells can only be kept in cell culture for a finite period of time, if at all. Therefore, you often need to obtain primary cells directly from an animal source. After which, you may fix and image the primary cells in situ (as part of the whole organ), or you may isolate individual primary cells for fixation and individual imaging.

Whatever your primary cell imaging goal, below you will learn how to both fix in situ primary cells and isolated primary cells for great imaging results.

How to Fix In Situ Primary Cells (Whole Tissue)

1) Collect

Harvest your tissue of interest and immediately mince your tissue into 2 mm3 cubes using a sterile scalpel or scissors. Rinse tissue a few times in chilled PBS* to remove any contaminates (hair, paper towel, etc).

*PBS or phosphate buffered saline is a common buffer used in cell culture and histology. The osmolarity and ion concentration of PBS is the same as human tissues, so it will not cause your cells to shrivel or explode. PBS is 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, and 2 mmol KH2PO4 at pH7.4. Be sure to sterile filter your PBS.

2) Fix

Immediately following harvesting, you will need to fix your cubed tissue samples to minimize degradation. Therefore, have your fixation solution aliquoted, labelled and on ice, ready to go before you sacrifice any of your animals. There are two common ways to fix your tissue: organic solvents and cross-linking. There is no way to anticipate the best fixation method for your staining or immunohistochemistry needs. Instead you will likely need to test a variety of fixation conditions for your particular situation.

Organic solvents method: In this method organic solvents such as alcohol or acetone are used. These organic solvents work to preserve your samples by removing lipids, dehydrating your tissue, and denaturing and precipitating the proteins in your cells. To fix with organic solvents, fully submerge your tissue cubes into ice-cold methanol, ethanol or a 1:1 mix of ethanol and methanol, and incubate your tissue in this solution in the freezer (-20°C) for an hour.

Cross-linking method: In this method paraformaldehyde is used to form covalent chemical bonds (or cross-links) between the proteins in your tissue and their surroundings. This method usually provides the best preservation, especially of soluble proteins, but it can also ‘mask’ your antigens. If your antigens are ‘masked’ this means that the cross-linking is preventing your antibody from recognizing your antigens. See Catriona Paul’s article ‘Antigen Retrieval Techniques for Immunohistochemistry: Unmask that Antigen!’ for unmasking protocols. To fix by cross-linking fully submerge your tissue cubes into freshly prepared 4% paraformaldehyde/PBS* solution. Incubate your tissue in this solution for 30 minutes at room temperature.

Note: You can fix tissue samples larger than 2 mm3,but they require longer fixation times and this can lead to fragile tissue which shatters when sectioning. If you want to fix larger samples be prepared to play around with your incubation times and conditions to find what works best. Your goal is to have tissue that is fixed and has well preserved structures, but is not too brittle to section.

3) Rinse and dehydrate

When you are done fixing, rinse your tissue sample a few times in PBS* to remove any remaining fixative. After this, the tissue is ready for processing and embedding, but this is a subject too large to also cover in this article! Don’t worry though- it’s coming next month!

How to Fix Isolated Primary Cells

You can purchase some primary cell lines from companies, but often you will have to isolate directly from a tissue source. As primary cells are thought to be more biologically relevant than immortalized cell lines, the hassle of harvesting and isolating your own primary cells is sometimes worth it. To harvest, you need to disrupt the extracellular matrix holding the tissue together to liberate the cells. This is most commonly done by using a proteinase, such as trypsin. Some call this process ‘enzymatic disaggregation’ to which I say, “They are making it sound way more complicated than it is!”. To liberate primary cells from tissue (using trypsin) is relatively straightforward:

1) Collect tissue

Harvest your tissue of interest. You want to work as fast as possible here to minimize cell degradation. Mince your tissue into 3 to 4 mm3 cubes using a sterile scalpel or scissors. Rinse your tissue a few times in chilled PBS* to remove any contaminates (hair, paper towel, etc).

2) Digest

Immediately plop your tissue into your cell dissociation solution. Now, what type of cell dissociation solution you use is a matter of opinion. There are lots of commercial cell dissociation agents on the market but you can also just use a sterile 0.25% Trypsin, 1 mM EDTA, PBS* solution.

3) Agitate

Agitate your tissue/cell dissociation solution until your tissue disintegrates. How long you agitate is a balancing act! The longer you agitate, the higher your cell yield will be but the more likely your cells will be damaged. So you may need to try a range of times until you find an incubation time that gives you an acceptable yield of viable cells.

4) Collect cells

After you have successfully dissociated your tissue, your cells need to be collected by centrifugation (~2,000 rpm for ~5 minutes should do it). After your cells are collected at the bottom of your tube, aspirate off the used cell dissociation solution. Now you need to rinse your cells a few times. To rinse, re-suspend your cells in PBS*, centrifuge, aspirate off the old PBS* and repeat 2 to 3 times. These rinsing steps are important to remove any remaining trypsin and stop the digestion.

5) Sort (if needed)

Depending on what kind of primary cells you are after, you may then need to do some sort of sorting by size or by FACS before proceeding.

6) Fix isolated primary cells to cover slips

Once you have isolated the primary cell type you are interested in, you can fix them and then smear them directly onto your microscope cover slips, using centrifugation, just like you would with a regular immortalized suspension cell line. See my article on fixation of suspension cells. Or you can culture your primary cells and treat them as you would a regular immortalized adherent cell line by growing them on cover slips then fixing- see my article on fixing adherent cells.

7) Unmask

There are a variety of unmasking techniques out there. Most of which use some combination of proteinases, heat and chelators to make your cells more easily penetrated. See Catriona Paul’s article ‘Antigen Retrieval Techniques for Immunohistochemistry: Unmask that Antigen!’ for unmasking protocols.

8) Stain and image

When done fixing and unmasking (if needed), your cells are ready for staining and imaging when you are. However, if you are not ready just yet, your cover slips with their fixed cells can be stored under PBS* in the refrigerator for up to three months.

Good luck and happy fixing!

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Image Credit: TheJCB

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