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Methods in Cell Biology

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1 Methods in Cell Biology
Medical Microanatomy 602 Edie C. Goldsmith, Ph.D.

2 Introduction The material in this power point is covered in Chapter 1 of your textbook. I would encourage you to read the text, particularly sections that are covered here. While we will not specifically cover this material during lecture, you will be expected to know this information for the exam. I can address any questions you may have during the lab period or by . Notice that in the notes section below each slide I have added information and explanations for the images/text on that slide when necessary.

3 When you look at these images what do you see
When you look at these images what do you see? Most of you, if not all, will probably recognize that the image on the left is roughly half of a heart while the image on the right is a small slice (a section) of heart tissue. What other information can we get from these images? At this point, not much. We can’t tell how many different cells types are present in the heart from these pictures or how they are organized. We can’t see if there is any material between these cells which might play a role in how the heart functions. In order to see these details, there are certain things that must be done to this specimen or section. This presentation will describe various histological methods which are used to visualize details in cells/tissues/organs that are important for their normal architecture and function.

4 Everything starts with the specimen, in the case of our previous slide that would be a heart. Don’t get caught up in memorizing all the steps in this chart. You need to recognize that no matter which type of microscopy the sample is being prepared for (light or electron microscopy-EM) there are certain common steps (noted by the light blue arrows on the right) in sample processing. These are fixation, embedding (not necessary for all cases), sectioning, mounting and staining. In the next couple of slides, we will look at these common steps in more detail.

5 Tissue Preparation Fixation Not all fixative works for all structures
Preserve tissue structure Chemical Formaldehyde/glutaraldehyde - cross-linking amine groups Alcohol – denature proteins Heavy metals (osmium tetraoxide) – oxidation of proteins to form cross-links; also react well with phospholipids Freezing Cryoprotectant (OCT, sucrose solution) Rapid low-temp freezing Lipids and activity stains (enzymes) Not all fixative works for all structures Formaldehyde - not good for lipids Don’t over fix The first step in tissue/organ preparation is fixation. Fixation serves to stop metabolic processes and prevent degradation of cellular structures. Fixation can be done using chemicals (common ones are listed above as are their mechanisms of action) or by freezing. Freezing is usually used as a fixation method when you are interested in a molecule (enzymes for example) whose activity would be destroyed by chemical fixation or that might be lost (in the case of lipids) due to sample processing. When using freezing it is important to use a cryoprotectant (i.e. OCT – optimal cutting temperature) in the freezing process so that ice crystals do not form in the tissue (these could lead to fixation artifacts and loss of cellular structures) and to make sure that it is done rapidly (putting the tissue in the freezer of your refrigerator won’t do – most cases something like liquid nitrogen, -196°C, or isopentane, -80°C, is used). Frozen sections are often used during surgical procedures when the physician needs to determine if all of a diseased tissue has been removed (see Clinical Correlation Folder 1.1 pg 4). Since formaldehyde works by cross-linking proteins together through amines, why do you think this would be a bad fixative for lipids? (hint - think about lipid structure) You can get too much of a good thing. It is possible to over fix a sample. While this might not cause many problems if you are just going to stain the sections with dyes, if you want to look for the presence of a particular protein, say you’re looking for a tumor antigen, over fixation can alter the target protein enough that the antibody used to detect the protein will no longer recognize it.

6 Dehydration & Clearing
Prepare tissue for embedding Removal of water can cause tissue shrinkage Infiltration Mix of clearing agent and embedding media Embed tissue Prevents tissue damage during sectioning Embedding agents - Paraffin, plastic resins, acrylamide Section, mount and stain The vast majority of slides you will examine this year will be from paraffin embedded sections, so we will go over what happens next for these samples. Paraffin is a wax, and to prepare the specimen for embedding it must first be dehydrated (done by incubating the specimen in alcohol solutions with increased alcohol content – start with 50% or 70% ethanol going all the way up to 100% ethanol) and cleared. Clearing involves incubating the specimen in an organic solvent which is miscible with both alcohol and the paraffin. Since tissues have a substantial water content, removing the water can cause the specimen to shrink, producing artifacts (something that is not normally present in the tissue). However, as we will talk about later on some of these artifacts can be used to help us classify certain tissues. The specimen is placed in melted paraffin (the embedding medium) and the paraffin is allowed to cool and harden. The addition of a hot solution can also lead to the formation of artifacts in the specimen. Different embedding medias can be used (examples are given above) depending upon the application the specimens will be used for. Depending upon the embedded medium used, the specimen is then cut into thin sections (for light microscopy typical paraffin sections run 5-10 microns thick while acrylamide sections can be microns thick), mounted on a slide for support and stained.

7 Samples are dynamic during processing Some things get retained
Nucleic acid – protein complexes, cytoskeletal proteins, ECM proteins, lipid-protein complexes Some things get lost Small, soluble molecules (ions, carbohydrates), glycogen, proteoglycans Can use special fixation or staining methods It is important to keep in mind that while the overall appearance of a specimen doesn’t change during processing (refer back to the slide with the piece of heart tissue vs the heart section – you can still tell both of them are heart), the distribution and types of materials present within the cells may change. Large molecules, proteins for example, that won’t dissolve in the fixative/processing solutions or can react with other molecules to form large complexes will be retained within the cells. Smaller molecules, which are easily solublized, will be lost during sample preparation.

8 Tissue Staining Basophilic Stain with basic dye [dye+Cl-]
Toluidine blue, methylene blue, hematoxylin, alcian blue Nucleic acids, some cytoplasmic components (rRNA and rER), glycosaminoglycans and acidic glycoproteins Acidophilic Stain with acidic dye [Na+dye-] Orange G, eosin, acid fuschin Mitochondria, cytoplasm, secretory granules, ECM proteins Without staining, the tissue section would remain translucent and we would still have difficulty identifying the relevant cell types and features. Staining using colored dyes provides a mechanism for introducing contrast. Dyes can be roughly grouped into two categories, basic or acidic, based on the chemical nature of the dye itself. Basic dyes carry a net positive charge and tissues that stain well with these dyes are referred to as being “basophilic”. Some examples of basic dyes are shown in the left hand column. Since basic dyes have a positive charge, they are going to bind to cellular components with a negative charge (some examples are given). Do you know why basic dyes would bind to nucleic acids? (hint: think about their structure) There are three chemical reactive groups found on biomolecules which can react with basic dyes: phosphates, sulfates and carboxyl groups. These groups have different pKa’s so by changing the pH, one can use different basic dyes to identify different structures within a section. Acidic dyes carry a net negative charge and tissues that stain well with these dyes are referred to as “acidophilic”. Some examples are listed in the right hand column. Acidic dyes are much less specific than basic dyes, but stain a broader range of molecules within and around cells (examples of material which stain with acidic dyes are shown). Dye color does not determine whether a dye is acidic or basic (see Table 1.2 in your textbook)

9 Nuclei stained with hematoxylin Hematoxylin & eosin (H&E) is the most common dye combination you will see this semester. Hematoxylin – basic- like dye which stains negatively charged molecules (blue) i.e. Nucleic acids Eosin - acidic dye which stains positively charged molecules (pink) Cytoplasm (proteins) The image shown above is of skeletal muscle cut in two different planes. Material stained with eosin

10 Metachromasia When certain dyes bind some cellular structures, their color shifts This is due to the presence of a large number of polyatomic anions (SO42- and PO43-) This leads to aggregation of the dye which changes is absorption properties The polyatomic anions are common in ground substance (particularly cartilage); heparin granules in mast cells; rER in plasma cells Toluidine blue is an example of a dye the exhibits metachromasia. As it name would imply, this dye usually stains blue. But when it is used to stain mast cells, the granules in these cells contain lots of negatively charged molecules and the dye’s color shifts to a purple-red appearance.

11 Other notable stains Periodic acid-Schiff (PAS)
PAS positive Staining (mucus) Periodic acid-Schiff (PAS) Carbohydrates (glycogen, mucin, basement membrane) Periodic acid + sugar aldehyde aldehyde + bleached basic fuchsin (Schiff reagent) magenta color PAS reaction stains carbohydrates and carbohydrate rich molecules (examples are given above). You will also see a fair number of examples of this stain during the semester. Periodic acid reacts with hexoses or hexosamines to form an aldehyde group. The aldehyde then reacts with bleached basic fuchsin (also called a Schiff reagent) to produce a magenta color (see mucus in goblet cell in the above image). Note that the PAS reaction is not quantitative as compared to the Feulgen reaction described below. A similar reaction (Feulgen reaction) occurs when DNA is depurinated under acidic conditions (usually treatment with HCl). This leads to aldehyde formation as happens in the PAS reaction, but in this case reaction with the Schiffs reagent occurs stoichometrically, so the colored product can be quantified and this method can be used to determine the amount of DNA present.

12 Orcein Also known as resorcin-fuchsin
stains elastin found in elastic fibers The individual fibers in this image are elastic fibers stained with orcein Aorta

13 Trichrome stains Azan Employ 3 dyes Masson Trichrome– most common
Usually used to look at collagen in the extracellular matrix Nuclei (basophilic) – blue/black Collagen – green or blue Cytoplasm, muscle, keratin, erythrocytes – red Azan Stains collagen, basement membrane, mucin Nuclei – red Collagen - blue erythrocytes nucleus collagen The upper image on the right is an example of a Masson’s Trichrome stain and the lower one is a example of an Azan When I indicated under the Trichrome stain that the nuclei are basophilic, you know what that means right? nucleus collagen nephron

14 Picrosirius Red Collagen specific stain
Observed under polarized light the collagen appears yellowish-orange in color. Observed under normal bright field microscopy, the collagen would have a bright pink appearance. In this image of skeletal muscle, the collagen has a bright, glowing orange appearance. The striated structures are muscle cells.

15 Nissl This uses a basic dye on neuronal tissue.
Stains the rER in neurons Referred to as Nissl substance when observed in clumps Can also be done with methylene blue “N” in the above image of a neuron indicates the grainy appearance of Nissl substance in this cell (all of the grainy magenta material around the N is Nissl substance (rER); the black A refers to the axon. We’ll talk more about this when we discuss Nervous Tissue in a couple of weeks.

16 Gold and Silver Stains Renal cortex Gold staining of motor end plate
Sometimes metals can be used to stain certain features within tissues, like the examples shown above, although the chemistry behind these reactions is unclear. Renal cortex Gold staining of motor end plate in muscle Silver stain of reticular fibers (type III collagen) String-like appearance in this image

17 Lipids Preserved in frozen sections Stains used in light microscopy
Sudan black, Oil red O, Sudan IV, Osmium tetraoxide Common stain used for electron microscopy Osmium tetraoxide I noted in this slide that lipids were preserved in frozen sections. This is an example of when knowing what you are looking for in a tissue will dictate what type of fixation is used. What would this image look like if this tissue had been treated with a chemical fixative and embedded in paraffin? Would the lipid droplets be visible? Black circles in this image are lipid droplets in brown fat cells

18 Enzyme Histochemistry
Localize enzymes within tissue Don’t over fix (frozen sections) Precipitated product Indirect method Variety of enzymes Acid and alkaline phosphatases, dehydrogenases, peroxidases Light and EM This technique is used to localize enymes within a tissue. Again, fixation is key – if you over fix or use the wrong fixative you can destroy the activity of the enzyme. This is what is called an “indirect method” because what will be seen is a precipitated product from the enzyme reaction, not the enzyme itself. The method requires a cleavable substrate and a trapping agent (in the image above the trapping agent is lead), so that when the substrate is cleaved the trapping agent forms a precipitate with one of the end products. This technique can be used with a variety of enzymes (examples are noted above) and since many of the trapping agents used are heavy metals, these sections can be viewed by light or electron microscopy (EM). Alkaline Phosphatase in rat kidney (Gomori stain with a lead precipitate)

19 Immunohistochemistry (IHC)
Rely on the use of antibody directed against molecule of interest Usually protein Direct vs indirect Amenable to many forms of detection Fluorescence EM (gold) Light microscopy (color precipitate) This technique is commonly used to localize proteins (or some small molecules) to certain cells within a tissue section. It relies on having an antibody against the molecule of interest (this is called the primary antibody) and can either be a direct or indirect method. In this case, direct vs indirect refers to the detection method. In a direct IHC, the reporter (fluorescent dye, gold, enzyme) is directly attached to the primary antibody. In an indirect IHC, the reporter is attached to a secondary antibody which will bind to the primary antibody. The advantage of the indirect approach is that you can bind multiple secondary antibodies to a primary antibody, thus increasing the sensitivity of the assay (see Figure 1.5 on page 9 in your textbook for a cartoon describing this). Blue material in this fluorescent image is a cell surface protein that binds collagen.

20 a-amylase ab stained with Protein A - gold
On the left, you have an example of IHC in which the primary antibody (abbreviated as ab in the caption) was against a-amylase and the reporter in this case is gold bound to Protein A (Protein A is derived from bacteria and binds antibodies). This image was collected by EM. On the right is an IHC in which the primary antibody was aginst lysozyme and the enzyme peroxidase was attached to the secondary antibody. For enzyme detection, a substrate is added which in the presence of the enzyme (in this example peroxidase) is converted into a colored, precipitate (orange shown here). a-amylase ab stained with Protein A - gold Orange indicates sites of peroxidase reaction (lysozyme in small intestine)

21 Parathyroid hormone staining (red-ish color) in
In Situ Hybridization DNA or RNA distribution Target specific nucleotide probe Radioisotope, biotin, digoxigenin Detected by autoradiography, color precipitate, fluorescence This method is similar in concept to IHC, but instead of looking for a protein you are detecting DNA or RNA. Instead of using an antibody, a small oligonucleotide which is complementary to the target DNA/RNA sequence is used. The oligonucleotide (either DNA or RNA) can be labeled with small molecules (biotin, digoxigenin) or a radioisotope (32P, 35S). If a radioisotope is used the target can be observed by autoradiography. If a small molecule is used, detection can be accomplished with either an labeled antibody (for digoxigenin) or another biomolecule (labeled streptavidin in the case of biotin – streptavidin binds specifically to biotin with very high affinity). Parathyroid hormone staining (red-ish color) in Parathyroid gland

22 Fluorescence Microscopy
Light source (UV or laser) Dyes which when excited emit in the visible region of the spectrum Confocal microscopy Laser light source Scan across sample and optically section Useful on live samples (monitor real time events) Non-destructive (in that it can be used on live cells) Easy sample preparation, fast, inexpensive In cases where an antibody is labeled with a fluorescent dye or the detection reagent is fluorescent, fluorescence microscopy is used. Confocal fluorescence microscopy is commonly used to examine the organization/distribution of biomolecules within cells. This technique can be used on live or dead samples, and because the laser can be moved (scanned) across the specimen as well as down into the specimen, it can produce 3D images. The next slide shows a couple of examples of fluorescent images.

23 Not all fluorescent stains require an antibody
The images here demonstrate that in addition to using antibodies to visualize molecules of interest, we can also use some dyes which will bind biomolecules (such as acridine organe which binds DNA/RNA) or we can use fluorescently labeled proteins (that aren’t antibodies) such as phalloidin, a mushroom toxin, that binds to actin. Phalloidin staining of actin Acridine orange DNA – yellow; RNA - orange

24 Autoradiography Rely on the use of radioactive tag to follow molecule
32P, 35S, 3H Radioactive probe (amino acids, nucleotides) Specimen must be alive to monitor metabolic events Can be used with other stains Slide dipped in photographic emulsion & developed Silver grains where radioactive material is present in sample (AgBr Ag (precipitate)) Semi-quantitative Autoradiography can be used to follow the fate of a biomolecules, but requires the introduction of a radiolabeled pre-cursor (amino acid, nucleic acid) into a living system (be it cells in culture or a whole animal). This technique has been use often in research to monitor the production/localization of biomolecules. This method can be semi-quantitative in that one can count the number of silver grains present on the image to determine how much of the biomolecule of interest is present.

25 This is a low magnification light microscopy image (upper image) and high magnification EM (lower image) of the submandibular gland of a mouse that was given 3H-fucose. The dark dots in the upper image are the radioactive material which in the lower image appear as coiled structures present ether outside of the cells in the lumen (denoted “L”) or over granules (G) within the cells.

26 Electron Microscopy Electron beam as source (l ~ 0.005 nm)
Operate under vacuum Electromagnets instead of glass lenses Detect by fluorescent screen or photographic emulsion Destructive Two major types Transmission (TEM) and scanning (SEM) Electron microscopy provides the highest resolution of subcellular structures. Many electron microscopes are operated under vacuum, so they are not suitable for examining living specimens. The principles of EM are similar to light microscopy except that – EM uses an electrons as its source instead of light and electromagnets to focus the electron beam instead of glass lenses used to focus light in a typical microscope.

27 TEM Cellular ultrastructure Bright portion – e- pass through
Dark portion – e- absorbed or scattered Sections – 50 – 150 nm Fix – glutaraldehyde Stains (e- dense; heavy metals) Osmium tetroxide (OsO4) – lipids Uranyl acetate, Lead citrate – non-specific (surface adsorbed) Ruthenium red – complex carbohydrates Transmission Electron Microscopy (TEM) is great for viewing subcellular organelles. The set up of a typical TEM microscope is shown in the image on the right. Keep in mind that when you look at TEM images, and we will look at a number of them this week, the bright areas of the images are unstained (the electrons passed through the sample) and the darker regions are areas which have taken up stain and either absorbed or scattered the electrons.

28 SEM Surface ultrastructure
Fixed, dried, coated with gold before imaged e- reflected from surface Results in 3-D like image Scanning electron microscopy (SEM) provides information about the surface of a specimen, therefore these samples are not sectioned prior to imaging but are coated with a gold-carbon film. The electron beam is then scanned across the specimen surface and the electrons that are reflected off of the surface are captured by the detector. The resulting image has a 3D appearance. The setup of a typical SEM is shown in the cartoon.

29 SEM image TEM image Both of these images are of podocytes surrounding capillaries in the kidney, but provide vastly different information. The SEM on the left shows in tight juxtaposition of cell processes from the podocytes (indicated by the arrows) while the TEM shows cross-section of these processes as well as the nucleus of the podocyte.

30 Interpreting EMs Use internal rulers as guides
Plasma membrane ~ 10 nm wide Nucleus between 3-10 mm Ribosomes between nm Unique organelle features Mitochondria and Golgi Look for largest objects first When we start looking at EMs, resist the temptation to try and identify every last object in the image (you can’t). Instead, look for the largest, most distinctive objects first and use the relative sizing to help you identify subcellular structures. These will usually be the bigger organelles (nucleus) and the mitochondria, golgi or rough endoplasmic reticulum (rER).

31 Problems & Pitfalls Shrinkage – fixation and embedding
Appearance of empty spaces Empty spaces due to loss of material Improper fixation and dehydration Wrinkling Precipitation of stains (may appear as dark spots all over the section) Do not memorize images - learn morphological criteria Do not rely on color I mentioned that sample processing can lead to formation of artifacts within a sample. Keep this in mind when looking at images. Shrinkage can lead to the appearance of “empty space” between cells or you can loose material (such as lipids) during processing. If you see a perfectly round empty space that is smaller than the nucleus of the neighboring cell, think about what you are looking at and the relative sizes of organelles before you call this the lumen of a blood vessel. If you see a line of intense, dark staining that runs through a significant part of the section that could just be a wrinkle in the tissue section that developed when the section was mounted on the slide. Two final things: don’t memorize images, learn the criteria for identifying structures. For your exam, I will do my best to show you images that I haven’t used in class before so spend the time learning what a mitochondria looks like and how you identify one, not memorizing the images I showed you in class or lab. Don’t rely on color as a means to identify something. As dyes get old, their color can change but the structure of an organelle or the organization of cells within a tissue won’t. Again, spend your time learning the criteria of the subcellular structures and tissues that we will cover.

32 Millette’s Laws Law #1 – In any given image, if two objects look the same they are the same. Law #2 – In any given image, if two objects look different they are different. Students often want to compare the way things look from one slide to another (particularly if they are relying on color) and based on that try to identify them. Due to differences in staining, that is not a good idea. Use these two rules to help you know when you can use comparisons to help you.

33 Goblet cells in Intestine
Here are two different stains of goblet cells in the intestine. You can see the obvious difference in the ability to see mucus within these cells based on the stain of choice (What does PAS stain?) and how the samples were likely processed (Why are there clear areas like the one at the tip of the arrow? What should be there?) PAS H&E

34 A Tale of Two H&E Stains Both of these images are from sections of kidney stained with H&E. As you will learn later in the semester, despite the differences in color, the cells are organized the sample way in both of these sections. This is a great example of how important learning the morphological criteria are for identify structures/tissues/organs.

35 2-dimensional appearance vs. 3-dimensional structure
One of the most difficult concepts is that when you are looking at a section on a slide, it is a 2D image of a 3D object. As shown in the example above, you can cut the orange or kidney (shown in the H&E images) in different planes and while the images look slightly different (more of less of certain structural features are visible) it is still an orange or kidney. Think about how 3D objects would appear if they are cut differently and keep this in mind as you are looking at slides (How would a blood vessel appear if it were cut in cross-section versus a longitudinal view?).

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