Protocols
Protocols
- Tissue Processing for Light Microscopy
- Tissue Processing for Transmission Electron Microscopy
- Frozen Sectioning
- Special Stains
- Immunohistochemical Localization
- Tissue Sectioning for Electron Microscopy
- Interpretation of Morphology
- Reagents for Use in Morphology Projects
- In situ Perfusion Technique for Rodents
Tissue Processing for Light Microscopy
The best preservation of morphology requires adequate fixation. For most tissues, this means exposure to fixative as quickly as possible. Some protocols require whole animal or organ perfusion by fixative. Others tolerate quick dissection and then immersion in fixative. For immersion, the size of the tissue block is important; it should not be thicker than 1 cm. Also, for immersion, 15-20 volumes of fixative solution is necessary for each volume of tissue. Keep the tissue in fixative until it is ready to cut.
Only flat-bottom vials or containers should be used for fixation of the block of tissue. This allows the fixative to cover all sides. (Floating blocks require covering by a piece of paper towel.) The tissue also retains it shape as it fixes when resting on a flat surface. Do not use conical tubes for tissue fixation, as these deform the block.
Often it is necessary to trim the fixed block of tissue to provide a flat face for sectioning. With a steady hand, use a scalpel to make a clean cut across the surface you want to see microscopically. (No sawing, please!) Return the tissue to the container with the fixative.
Fixed tissue subsequently can be frozen or embedded in paraffin for sectioning for light microscopy. Specially fixed tissue can also be embedded in plastic for thin-sectioning for transmission electron microscopy.
In our experience, the following are adequate fixatives for usual research applications requiring light microscopy:
Basic Histology
Tissue to be stained using standard histological stains, need only to be fixed in neutral buffered 10% formalin which is a commercially available product from Fisher or Baxter. Formalin is extremely stable and can be stored at room temperature for an extended period of time. Tissue may be left in formalin indefinitely prior to processing for sectioning and staining.
Immunostaining
Immunostaining may be done on tissue which is either fresh frozen or fixed frozen as well as paraffin embedded, depending on the antibody. (Freezing techniques will be discussed in another section.) Fixation for immunostaining requires the use of 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. Time of fixation varies with the aldehyde sensitivity of the antibody, but 4 hours to overnight is customary. This fixative needs to be fresh from stock solutions of paraformaldehyde and phosphate buffer. While the stock buffer is stable if kept refrigerated, the paraformaldahyde stock is not. It should not be used after being stored in the refrigerator one month.
For your convenience, the recipes for buffers and fixatives are listed in reagents.
TUNEL Staining
Localization of apoptotic cells may be performed on fresh frozen or 4% paraformaldehyde-fixed tissue. Again, the immunoperoxidase method is used, and a light cytoplasmic counterstain is a matter of preference.
Tissue Processing for Transmission Electron Microscopy
Adequate preservation for ultrastructure requires immediate fixation. This usually means a quick dissection just after sacrifice. Then, only the tissue of interest must be rapidly minced into tiny pieces no larger than 1 mm3 to insure proper penetration of the fixative. Do this mincing with a scalpel in a drop of fixative. Make sure not to crush the tissue as you cut it. Once minced, the tissue should be placed in a vial of fixative
A variety of fixatives and buffers have been used for electron microscopy, and each has its advantages and disadvantages. One thing is certain: it is extremely important that all reagents for EM be made fresh.
In our experience, the following are adequate fixatives for usual research applications requiring electron microscopy:
- Routine Electron Microscopy--phosphate-buffered 2.5% glutaraldehyde
- Immunoelectron Microscopy--combination of paraformaldehyde and glutaraldehyde
For your convenience, the recipes for buffers and fixatives are listed in reagents.
Since fixation varies with certain structures or types of tissue, it is best if you contact the Research Morphology Core Facility prior to embarking on an EM project, so that we can discuss the best recipe for buffer and fixative for your particular tissue.
Frozen Sectioning
In order to section the tissue thin enough for light microscopy, the tissue must be either paraffin-embedded or frozen. Tissue for freezing can be either fresh or fixed, often depending upon the antibody to be used. In general, fixed tissue is immersed in 30% sucrose to cryoprotect the tissue. Recipes for buffers and fixatives are listed in reagents. If "freeze artifact" remains a problem in the histology, we will advise you regarding acceptable alternative methods. Then, the tissue is mounted using [OCT] (American Master Tech). Frozen 10 micron sections for immunostaining are placed on SuperFrost Plus glass slides from Fisher.
The Research Morphology Core Facility will be responsible for frozen sectioning for either on-slide or floating section staining. Regrettably, there is no possibility for time-sharing of our cryostat.
Special Stains
Hematoxylin and eosin is the stain best suited to routine histology. It works well on either frozen or paraffin-embedded tissue and stains nuclei dark blue and everything else various shades of pink. Once investigators are used to the appearance, interpretation is straightforward.
A variety of commonly used special stains is offered by the Research Morphology Core Facility:
| Tissue Type | Stain | Usage |
|---|---|---|
| Calcium | Von Kossa | Calcium appears black, noncalcificed, tissues = pink |
| Collagen | Masson’s trichrome | Collagen is blue or green, tissues = red or grey |
| Movat’s pentachrome | Elastic, collagen and noncollagenous tissues are distinguished. | |
| Picrosirius red | Collagen appears red and cytoplasm yellow. | |
| Elastin | Van Gieson | Elastic tissue is highlighted in black, collagen = cerise. |
| Fat | Oil red-O | Lipid is red-orange against a pale grey background. |
| Neurons | Luxol fast blue | Myelins stains blue-green |
These stains work well on either frozen or paraffin-embedded tissue. For pricing of these and other special stains, refer to the fee schedule.
Immunohistochemical Localization
Immunohistochemistry refers to the use of antibodies to label specific proteins within tissue sections. The Research Morphology Core Facility uses either commercially available antibodies or ones from private stock, however the latter should be fully developed with some sort of track record, including titration curve and known concentration for optimal results. Positive control tissue is essential. Although our laboratory will titrate reagents and adjust conditions for the investigator's particular tissue within reason (charged by the number of runs), we cannot undertake extensive evaluations of new, untested antibodies.
Because it is so dependent upon tissue epitopes and experimental conditions, immunohistochemistry has evolved into a subspecialty within morphology. The range of tissues and antibodies used on them means that there is a tremendous breadth of technique. Thus, the investigator should be knowledgeable regarding the proposed usage of the particular antibody of interest in tissue and be able to provide references from the literature, if necessary. In other words, there should be a certifiable basis for feasibility prior to trying immunohistochemical localization.
Fixation for Immunohistochemistry
Adequate fixation is paramount. Generally, fixation of the tissue in fresh 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, is standard. Recipes for buffers and fixatives are listed in reagents.
Some aldehyde-sensitive antibodies work only on snap-frozen fresh tissue. A few antibodies will work on paraffin-embedded tissue, often only after antigen retrieval. The tissue sections are placed on SuperFrost Plus glass slides from Fisher. These expensive slides are superior for tissue adherence during conditions required for immunohistochemistry.
For immunohistochemical localization, the Research Morphology Core Facility usually employs the peroxidase method. This technique provides readily discernible histology in permanent slides. Use of different chromogens enables double labeling, however the labor in these protocols increases exponentially. Immunofluorescence also is available as a quicker alternative to either protocol.
Tissue Sectioning for Electron Microscopy
Electron microscopy is very labor-intensive, and only a couple of the tiny pieces should be thin-sectioned for examination. Out of a vial containing perhaps 10 cubes of tissue, several representative pieces will be processed and embedded in Epon. A two-micron thick section of each will be prepared and shown to the investigator at the light microscope. The most promising one or two blocks will be further trimmed to include only the area of interest (perhaps 0.3 mm2), before thin (600 Angstrom) sections are made. Thus, it follows that electron microscopy provides a very narrow view of tissue ultrastructure, and projects must be designed accordingly.
Interpretation of Morphology
Investigators who submit properly prepared tissue will receive a high quality product. Yet, the results depend upon the interpretation of these data. Investigators and their trainees are in the best position for interpreting their own slides, however there is a learning curve for each project. Dr. Philip Ursell, an experienced pathologist, is available for consultation, if necessary. For example, issues regarding necrosis, inflammation or malignant change may require his input initially. Similarly, immunoperoxidase methods may be new to some investigators, and questions regarding signal vs. background may arise.
Reagents for Use in Morphology Projects
Fixatives
Paraformaldehyde Fixative
- distilled H2O
- paraformaldehyde, purified ("prill")
- 10N NaOH
For 500 ml of 8% buffered paraformaldehyde (pH7.3) stock solution:
Heat 300 ml distilled H2O to 60oC. Put on stir plate. Add 40 gm prill and stir for at least one- half hour. Add 10N NaOH by drops until the solution clears. Filter to another container, and bring volume to 500 ml using distilled H2O at room temperature. Store at 4oC.
To make 4% buffered paraformaldehyde, add equal volumes of 8% stock and 0.2M phosphate buffer. Use fresh.
Paraformaldehyde/glutaraldehyde Fixative
- distilled H2O
- paraformaldehyde, purified ("prill")
- 50% glutaraldehyde
- 10N NaOH
For 1 liter of 8% para/5% glut fixative:
Heat 700 ml distilled H2O to 60oC. Put on stir plate. Add 80 gm prill and stir for at least one-half hour. Add 10N NaOH by drops until solution clears. Filter into another container, and bring volume to 900 ml using distilled H2O. Add 100 ml 50% glutaraldehyde and mix.
To make 4% buffered para/2.5% buffered glutaraldehyde fixative, add:
- 1000 ml 8% para/5% glut
- 1000 ml 0.2 M phosphate buffer
- Adjust pH to 7.4. Chill to 4oC before using, and use fresh.
Bouin's Fixative
- 75 ml saturated picric acid
- 20 ml 37% formaldehyde
- 5 ml glacial acetic acid
Buffers
0.1M Phosphate Buffer pH 7.3
- 2.6 gm NaH2PO4.H2O (monobasic)
- 11.5 gm Na2HPO4.7H2O (dibasic)
- 1 liter distilled H2O
- Mix while stirring.
0.2M Phosphate Buffer pH 7.3
- 5.25 gm NaH2PO4.H2O (monobasic)
- 23 gm Na2HPO4.7H2O (dibasic)
- 1 liter distilled H2O
- Mix while stirring.
0.1M Phosphate Buffer pH 7.3 with 30% Sucrose for Cryoprotection
- 2.6 gm NaH2PO4.H2O (monobasic)
- 11.5 gm Na2HPO4.7H2O (dibasic)
- 300 gm sucrose
- 1 liter distilled H2O
- Mix while stirring.
In situ Perfusion Technique for Rodents
Whether you use a pump to perfuse, gravity flow or do it by hand by syringe, you will need a blunt-tipped (filed) needle, phosphate buffered saline (PBS) and freshly prepared 4% paraformaldehyde (for tissue immunostaining) or 10% formalin (for usual histochemical staining). The animal should be adequately anesthetized prior to starting the thoracotomy.
- Following thoracotomy, use a blunt-tipped butterfly (or needle) to pierce the pericardium and enter the apex of the left ventricle. Advance the needle along the septum, and cannulate the ascending aorta. To stabilize the needle, clamp the apical entry point around the shaft. Begin the PBS flow and snip off the tip of the right atrial appendage. Gently flush with approximately 50-100 ml to remove most of the blood from the circulatory system of the rat (25-50 for a mouse). The outflow from the atrium should become clear. If you are using syringes, carefully exchange the syringe, leaving the cannula in place. Avoid introducing air bubbles that can cause flow problems. If you are using a peristaltic pump, carefully remove the tubing from the PBS bottle and lower it gently into the fixative reservoir bottle. With a gravity flow apparatus, air bubbles usually are not a problem.
- Begin perfusing with the fixative. If you are perfusing from syringes, have several loaded and ready to be attached to the cannula when needed. You will need about 400-500 ml of fixative for a rat (200-250 for a mouse). Gently perfuse over about 10 minutes. The animal should become very stiff, and the outflow should almost cease when the animal is fully perfused.
- As soon as the animal is well-perfused, dissect your tissue, and transfer it to a flat-bottom container with the same fixative. Immersion fix for another 2-4 hours.
We cannot cut fresh human tissue. We also cannot provide image analysis, radioactive assays or processing for scanning electron microscopy.
Offered at Other Facilities Affiliated with UCSF Department of Pathology:
Confocal Microscopy , Stereology , Transmission Electron Microscopy, including Immunoelectron Microscopy
- Juan Engel, PhD
- VA/NCIRE Microscopy and Advanced Imaging Core
- juan.engel@
ucsf.edu - 415-221-4810 ext. 2625
Flow Cytometry
- Shuwei Jiang, PhD
- shuwei.jiang@
ucsf.edu - 415-514-3138