HISTOCHEMISTRY Objectives: 1. to become acquainted with specific staining techniques for cell components 2.

to think about how the chemical components of cells allow their identification 3. to re-familiarize yourself with the microscope This laboratory introduces specific staining techniques for identifying cell types and cell components. The fundamental problem a cell biologist must overcome when viewing cells with a microscope (other than small size!) is the lack of natural contrast. This problem is to be expected as living things are mainly water and offer only a few contrasting features. Chloroplasts and cell walls in plants are an exception. It is desirable for a staining technique not only to allow for taxonomic identification of cell types and cell features (i.e. the Wright's stain), but also to give information about the chemical makeup of a given cell (i.e. the periodic acid-Schiff [PAS] carbohydrate stain; Feulgen's reaction for DNA). Cell Types in Blood: Blood contains erythrocytes, leukocytes, and platelets suspended in plasma (See Table 1 below for more details). The erythrocytes, or red blood cells are the most numerous cell type in blood. These cells carry hemoglobin in the circulation and this protein transports oxygen from the lungs to tissues. Mammalian red cells are formed in the bone marrow of the adult by a process called erythropoiesis and then lose their nuclei shortly before they are released into the circulation. In contrast, erythropoiesis in nonmammalian vertebrates does not involve nuclear loss. Therefore, erythrocytes in the circulation of adult birds, reptiles, amphibians, and fish contain nuclei. In this lab you will prepare blood smears and stain for specific cellular components.

Blood Cells Type of Blood Cell Red Blood Cells (RBC) or Erythrocytes Granulocytes (Neutrophils = polymorphonuclear leucocytes) Granulocytes (Eosinophils) Main Function Transport O2 and CO2 Phagocytose and destroy invading bacteria Destroy larger parasites and modulate allergic inflammatory responses Typical Concentration in Human Blood (cells/L) 5 X 1012 cells/liter 5 X 109 cells/liter

2 X 108 cells/liter

Granulocytes (Basophils)

Release histamine (and in some species serotonin) in certain immune reactions Become tissue macrophages that phagocytose and digest invading microorganisms and foreign bodies as well as damaged and senescent cells Make antibodies Kill virus-infected cells and regulate activities of other leucocytes Kill virus-infected cells and some tumor cells Initiate blood clotting

4 X 107 cells/liter

Monocytes

4 X 108 cells/liter

Lymphocytes (B Cells) Lymphocytes (T Cells)

2 X 109 cells/liter 1 X 109 cells/liter

Natural Killer Cells Platelets = cell fragments that arise from megakaryocytes in bone marrow

1 X 108 cells/liter 3 X 1011 cells/liter

Fixation: Prior to the use of cytologic stains, tissue samples must first be treated with a chemical fixative. Fixation has three functions: (1) to kill cells and leave their internal structures in some approximation of the natural state; (2) to render cell components water-insoluble, and so, less likely to leach out of the tissue during subsequent handling, and (3) to facilitate the uptake of stains into the cytoplasm. There are a wide variety of fixatives that are categorized as to their ability to penetrate cells and preserve internal structures. Generally speaking, fixatives for light microscopy tend to be highly penetrating molecules such as methanol, ethanol, and formaldehyde. Fixation is the most critical step in any cytologic procedure. This step is often affected by a number of variables such as pH, temperature, osmotic balance, and duration of fixation. All of these elements must be empirically investigated when any new cellular material or system is to be examined. Wright's Stain: Histologists frequently use Wright's stain for the identification and differentiation of human blood cells. This stain is a mixture of methylene blue, methylene azure, and the eosinates of both. The azures act as bases and stain the basophilic (base-loving) components of a cell a blue color, while the eosins behave as acids and stain the acidophilic (acid-loving) structures of a cell a red color. Since there is a combination of dyes and a varying affinity for each, the various parts of the

cell are stained in hues of pink, purple, blue, and red. After Wright staining at pH 6-7, the structures in blood have characteristic colors, and each cell type is readily identifiable; the color patterns obtained are directly dependent on the pH of the staining reaction. The various human blood cell types as they appear in a fixed smear will be discussed. There are two classes of cells, red blood cells, or erythrocytes, and white blood cells, or leukocytes. The leukocytes are placed into two groups, the granulocytes, with conspicuous cytoplasmic granules, and agranulocytes, which lack them. The granulocytes include the neutrophils, eosinophils, and basophils. The agranulocytes include the lymphocytes and monocytes. In addition to these cell types there are the platelets, which are small cell fragments. Feulgen's Reaction: The Feulgen's reaction is a widely used test for DNA. Significantly, single-stranded RNA does not stain with this technique. Successful application of this procedure involves two steps. 1. Treatment of tissue with hot HCl. This acidic hydrolysis depurinates (removes purine bases) the strands of DNA, leaving a reactive aldehyde group at the (former) number 1 carbon of deoxyribose sugar. 2. Application of Schiff's reagent. Schiff's reagent (or leucofuschin) is prepared by treating basic fuchsin with sulfurous acid. When reacted with aldehyde groups, a reddish-purple dye is produced. Excess stain is rinsed from tissue using a bleaching solution of sodium metabisulfite to ensure that no unreacted leucofuchsin reverts to (colored) fuschsin. The Feulgen's reaction is widely used and produces excellent staining of nuclei. The chemistry associated with the Feulgen Reaction is shown below:

The details of the Leucofuchsin reaction with any available aldehyde are shown below:

PAS Reaction: This cytologic method is also based on the reactivity of Schiff's regent with aldehyde groups. In this case, aldehydes are produced in polysaccharides through periodic acid (HIO4) hydrolysis of vicinal hydroxyl groups. In particular, HIO4 breaks the 2,3-glycol grouping of saccharides, creating a dialdehyde. This reaction does not stain DNA or RNA, or any sugar that lacks the 2,3-glycol grouping. However, glycogen, starch, cellulose, glycolipids, glycoproteins, and many other carbohydrates in cells are stained. The PAS reaction is very effective as a plasma membrane stain, particularly in animal cells. The chemistry of the PAS reaction is shown below:

Prelab Questions relating to the background information (answer prior to coming to lab): 1. What is the major difference between mammalian and avian red cells? 2. Using the data in the Blood Cells Table above, calculate the volume in red blood cells in L. How does this calculated volume compare with your estimated volume from last weeks lab. 3. Which cell do you expect to be larger, the mammalian or the avian red cell? Why? 4. What is the purpose of the following steps in the staining procedures? a) fixation before staining b) blood smear treated with Feulgen's stain, but no HCl incubation c) depurination (heating in HCl) d) treating samples with bisulfite reagent (sodium metabisulfite) e) incubation with periodic acid 5. What chemical group does the Schiff reagent react with? 6. Why can Schiff's reagent be used for both Feulgen's and PAS reactions? +++++++++++++++++++++++++ Procedures: Each pair of students will prepare four blood smears - one with beef (or your own) blood and 3 with chicken blood. Make sure to smear blood on the frosted side of the slide. Preparing Blood Smears: Prepare blood slides as described in the diagram below:

Figure 1: Place 10 uL of blood at one end of a slide and PUSH the droplet with a second slide the length of the first slide.

The slides should be new, and unused, as rough edges will leave streaks on the smear. Slides should also be degreased in 95% ethanol so cells will adhere to the glass. It is important to move your hand rapidly when smearing the blood, and may be desirable to rest the hand on a table while making the smear. Also, if the blood on your prepared smear is not thin enough, you will not be able to see single cells. Dry the slides by waving them rapidly in the air. Make sure they are thoroughly dry before proceeding to the next steps.

Wright's Stain: For this stain, a fixation step is not needed since the stain is dissolved in methanol. 1. Using a wax pencil, label a beef or human blood-smear slide #1. (Include your initials for identification). 2. Lay slide on paper towels on your bench (frosted side or blood cell side up!) 3. Add enough Wright's stain to cover the smear (~1-4 drops); wait 2 minutes. 4. Add ~5-7 drops of 0.1 M K-Na phosphate buffer, pH 6.5, to the Wright stain: wait 4 minutes. 5. With forceps, dip the slide in distilled water to rinse (in a 250-mL beaker). 6. Place the rinsed, stained smear in a near vertical position to dry. 7. When dry, view the slide with a microscope. Immersion oil may be used directly on the blood smear, without a coverslip. 8. Capture an oil immersion image of the various cell types that you can find for inclusion in your results section.

Fixation Prior Feulgen's or PAS stain: 1. Fix three dried smears for 10 min in absolute ethanol-formalin (9:1). 2. Rinse the slides for 5 min in running tap water. 3. Rinse the slides briefly with distilled water.

Feulgen's Reaction: 1. Using a wax pencil, label two blood slides #2 and #3. 2. Place slide 2 into a Coplin jar labeled "1 M HCl", and incubate at 60 C for 10 min. (Slide 3 is a control and is not subject to acid hydrolysis). 3. Transfer HCl-treated slide (and the control slide) into the "Rinse" Coplin jar; rinse with a VERY gentle stream of tap water for 1 minute. 4. Hold slides with a forceps, and decant the tap water. Refill the jar with distilled water and let stand for 1 minute. 5. Transfer slides 2 and 3 to Schiff's reagent. Incubate the slides in the dark for 10 min. 6. At the fume hood prepare the "Bisulfite Reagent": to 180 ml distilled water, add 10 ml of 10% sodium bisulfite and 10 ml of 1M HCl. 7. Using forceps, transfer the slides to the "Rinse" jar that has been filled with distilled water. Rinse with distilled water for 10 sec. 8. Transfer the slides to the jar containing freshly prepared "bisulfite Reagent". Let stand 2 min. Decant and refill with new "Bisulfite Reagent". After 2 min, again decant and refill once more with new "Bisulfite reagent". 9. Decant again and irrigate (rinse) with tap water for 5 min. After 5 min, replace tap water with distilled water. Rinse briefly, then remove each slide; wipe the back of each slide with a Kimwipe & air dry in a near vertical position.

10. Counterstain in Fast Green for 2 min. Approximately 1/2 of the blood smear should be covered with staining solution. (This stain will bring out the color of the cytoplasm). 11. With forceps, transfer the slides to the "Rinse" jar that has been filled with distilled water. Rinse for 10 sec. 12. Remove each slide; wipe the backside with a Kimwipe, and air-dry in a near vertical position. 13. When the slides are completely dry, examine the cells under the microscope, using the high power and then the oil immersion objective. 14. Capture an oil immersion image of the Feulgen stained cells for inclusion in your results section.

PAS Method: Using a wax pencil, label one blood smear - #4. Place the smear in the HIO4 (periodic acid) Coplin jar for 10 min. Rinse as before with distilled water in the "Rinse" jar. Transfer the slide to Schiff's reagent. Incubate in the dark (foil-covered Coplin jar) at room temperature for 10 min. 5. While waiting, prepare "Bisulfite Reagent " as before: to 180 ml distilled water, add 10 ml 10% sodium bisulfite and 10 ml 1 M HCl. 6. Transfer slide with forceps into "Rinse" jar that is filled with distilled water. 7. Transfer slides to jar containing "Bisulfite Reagent". Let sit 2 min. Decant, and fill jar again with new Bisulfite reagent; let sit 2 min. Decant, and fill jar a third time with new Bisulfite reagent and let sit 2 min. more. 8. Decant and rinse 5 min with running tap water. Replace with distilled water; rinse briefly, then remove the slide. Wipe the back with a Kimwipe and air-dry in a near verticle position (cells = pink). 9. Place the air-dried slides in the "Hematoxylin" Coplin jar for 2 min (back bench Rm 209). Approximately one-half of the smear should be covered with staining solution. (Hematoxylin is a counterstain that binds all acidic components of the cell, including ribosomes and nuclear material. 10. Transfer slides to "Rinse" jar containing distilled water. Rinse for 10 seconds. 11. Remove slides; wipe back side with a Kimwipe and air-dry in a near vertical position. 12. When the slides are completely dry, examine the cells under the microscope, using the high power and then the oil immersion objective. 13. Capture an oil immersion image of the PAS stained cells for inclusion in your results section. 1. 2. 3. 4.

Study Questions related to your slides (to be completed during or after lab): 1. How many classes of cells can you identify with the Wrights stain? 2. Describe the classes of cells and their relative abundances of cells identified with Wrights stain. 3. Can you identify any organelles with Wrights stain? 4. Why is PAS an effective plasma membrane stain? 5. Based on your measurements of mammalian and avian red cells, how much larger (in percent) are avian red cells than mammalian red cells.? 6. How much larger are eosinophils than monocytes?

References Reuter, Larry (1995) WSU Cell Biology Lab Manual. Winona State University. Choinski JS (1992) Experimental Cell and Molecular Biology, Wm. C. Brown Publishers. Reid Philip D (1992) Tissue Printing: Tools for the Study of Anatomy, Histochemistry, and Gene Expression. Academic Press.

Ideas for Extension of this Lab:

Enzyme Cytochemistry: Enzyme cytochemistry is used to locate specific enzymes at their site of action within cells. The enzyme/substrate interaction results in an insoluble reaction product . There are entire manuals of stain recipes for enzyme/substrate reactions that result in an insoluble product that can be visualized by microscopy (or following transfer of cells and tissues to nitrocellulose, or the products may be localized in a gel). For example, there are stains for the insoluble products of the enzymes tyrosinase and peroxidase, (enzymes you will encounter later). You may wish to include enzyme cytochemistry as part of a more extensive characterization of an enzyme such as tyrosinase or peroxidase.

Histochemistry: Histochemical stains may be use to detect many different types of molecules, either directly on a slide or following transfer to a membrane called nitrocellulose (tissue printing). A few more examples of histochemical stains are: Ferric chloride - (to detect phenols, tannins, etc.) - 1% in H2O Toluidine blue - (to visualize anatomy) - 0.1% in water Methyl red - (to detect lignin in plants)- 0.01% in water I2 K I - (to detect starch) - 0.3g I2, 1.5 g KI in 100 mL H2O Starch KI - (to detect H2O2) 4% soluble starch 0.2 M KI Heat starch suspension to about 90% for 1 -2 minutes. Cool. Add KI to make a 0.2 M solution Cytogenetics: Rapidly-growing roots from onion bulbs may be soaked in various agents and analyzed for their effect on mitosis. Caffeine, for example, causes complete inhibition of mitosis, primarily through the inhibition of cell plate formation. Colchicine is a metabolite from the crocus plant which inhibits spindle fiber formation during mitosis. It can be added to the growing cells to halt cell division at metaphase. 8-Hydroxyquinoline is effective in contracting chromosomes for counting, and dyes such as Acridine Orange result in abnormal metaphases, isochromatid breaks, chromatid exchanges and anaphase bridges to name a few. Household compounds such as mustard, hot sauce, orange peel, tobacco, shoe polish have resulted in chromosomal damage in bacteria. These and others compounds may be worth testing on plant chromosomes.

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