Immu2011 Prac Notes Prac 1-5 PDF

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Summary

Prac 1 - Introduction to Immunology and Histology To understand what a microscope is and what they can be used for in immunology History of Microscopy First light microscope: 2 Dutch spectacle makers, 1950 Robert Hooke: coined the phrase 'cells' - perforates, porous, honeycomb, not regular World of ...

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Immu2011 Practical Summary notes Prac 1 - Introduction to Immunology and Histology •

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To understand what a microscope is and what they can be used for in immunology History of Microscopy First light microscope: 2 Dutch spectacle makers, 1950 Robert Hooke: coined the phrase 'cells' - perforates, porous, honeycomb, not regular World of Microscopy Used for pathology, oncology, haematology, microbiology, rheumatology, immunology Different types for diagnostic/research lab: Upright, Inverted, Phase-Contrast, Köhler & Fluorescence microscopes 3 Brightfield microscopes: Upright microscope: light from underneath Inverted: light source inverted ie comes from above Phrase-Contrast Kohler: evenly illuminated field of view, wide cone of light on specimen

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Other microscopes: Fluorescence microscopes: UV light excite fluorescent molecules on/in specimen

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In Immunology Pracs: mostly using compound brightfield upright microscopes Magnification of specimen compounded by magnifying effect (ocular and objective) Sizes of Objects Unit

Subunit

Example

1cm

10mm

Organs or their parts

1mm

1000um

Small organisms

0.1mm

100um

Tissue components

1000nm

1um

Cell organelles, bacteria

1nm

10Å

Molecules

0.1nm

1Å

Atoms

Cells and tissues

Dimension

Skin epidermis

100um-1.5mm

Intestinal Villi

1mm

Arteriole

30-200um

Hepatocyte (in the liver)

25um

Neutrophil

12um

Capillary (peripheral veins)

4-10um

Erythrocyte (red blood cell)

7um

Platelet

2-3um

Light microscope resolution

200nm

Plasma membrane

10nm

Immu2011 Practical Summary notes Light microscopes: resolution limited by wavelength of visible light spectrum (400650nm) Electron microscopes: obtain higher resolutions / smaller wavelengths eg UV (200400nm), technology constantly updating

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To be able to identify the various components that make up a microscope Structural Features of Upright Microscope

1. Eyepieces

14. Locking screw for condenser

Immu2011 Practical Summary notes 2. Binocular section of the tube 3. Binocular tube 30°/20 with integrated HD IP camera 4. Interfaces on reverse side of the binocular tube 30°/20 - for export of your images 5. Carrying handle 6. Control elements of the binocular tube 30°/20 7. Plug-in power supply 8. Illumination intensity display 9. Rotary knob for switching ON / OFF and setting illumination intensity 10. Focusing drive for fine adjustment (right side) 11. Focusing drive for coarse adjustment 12. Control knob for X travel of mechanical stage 13. Control knob for Y travel of mechanical stage

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15. Centering screw for condenser on condenser holder 16. Knurled ring for setting luminous field diaphragm (Full-Köhler equipment only) 17. Microscope stage 18. Abbe condenser 19. Objective 20. Knurled ring of nosepiece 21. Lever for adjusting condenser aperture diaphragm 22. Knurled ring for condenser height adjustment 23. Knurled ring for adjusting tension of coarse focus 24. Focusing drive for coarse adjustment (left side) 25. Focusing drive for fine adjustment (left side)

How to setup and use a microscope properly Adjust chair > appropriate height / distance Set interpupillary distance

Adjust viewing height

Correct interpupillary distance

Immu2011 Practical Summary notes

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Adjusting Transmitted Light Brightfield on Full-Kohler Microscope Enable optimal specimen viewing NOTE: The size of the field of view and the objective aperture change after every objective change. Therefore, repeat the adjustment of the luminous-field diaphragm and aperture diaphragm to obtain optimum results. Rotate the 10x objective into the light path using the knurled ring of the nosepiece (20 in the diagram). Place a high-contrast specimen slide with the coverglass being on top in the specimen holder of the mechanical stage (17 in the diagram). Fix the slide in place by means of the spring lever. Adjust illumination intensity using the rotary knob on the microscope stand (9 in the diagram). Move the Abbe condenser up to the limit stop using the knurled knob (22 in the diagram) and set the control lever of the aperture diaphragm (21 in the diagram). Set the eyepieces to "0" in line with the white dot on the rim of the eyepieces ( 1 in the diagram). If you wear prescription glasses and know what your prescription is, rotate this number (instead of "0") to be in line with the white dot. Look through one eyepiece of the binocular tube and bring the specimen into focus using the focusing drive (10 and 11 on the right side, or 24 and 25 on the left side in the diagram). Close the luminous-field diaphragm (16 in the diagram) until it becomes visible (even if not in focus) in the field of view (A in Fig. 6). Turn the knurled knob for vertical adjustment of the condenser (22 in the diagram) until the edge of the luminous-field diaphragm appears sufficiently sharp ( B in Fig. 6). Centre the image of the luminous-field diaphragm (C in Fig. 6) using both centring screws at the front of the condenser (15 in the diagram). Then, open the diaphragm until it just disappears from the field of view (D in Fig. 6). To adjust the aperture diaphragm (contrast), move the condenser aperture diaphragm (21 in the diagram) to match the objective magnification.

Figure 6: Adjusting the luminous field diaphragm.

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To understand the principles of immunohistology Identifying Immune Cells and Structures Human Blood Smear - Diff-Quick Stain

Mouse Secondary Lymphoid Organs - H& E Staining

Immu2011 Practical Summary notes

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Determine patient blood disorder Follows complete blood count abnormality, blood disorder symptoms eg jaundice, anaemia, abnormal bruising or bleeding, fatigue, bone pain, or fever Diff-Quick Stain: Giesma stain, identify subsets of present cells Cell Type

Colour

Erythrocytes

Pink/yellowish red

Platelets

Violet/purple granules

Neutrophils

Blue nucleus, pink cytoplasm, violet granules

Eosinophils

Blue nucleus, blue cytoplasm, red granules

Basophils

Purple/dark blue nucleus, violet granules

Monocytes

Violet nucleus, light blue cytoplasm

Lymphocytes

Violet nucleus, minimal cytoplasm

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Assess excised or biopsied tissue Quick and simple, general overview of tissue structure 2 stains 1. Haematoxylin - cell nuclei blue 2. Eosin - cytoplasm / EC matrix pink Some (irrelevant) variations in tissue/structure colour

Prac 1 Activity 2 Perform an image search or Pubmed search for examples of the following: A blood smear stained using A section of mouse A section of mouse spleen stained using lymph node stained Diff-Quick H&E using H&E

Immu2011 Practical Summary notes

Photomicrograph of Diff-Quik stained blood smear from black rhinoceros, Siabuwa infected with T. bicornis. https://www.researchgate.net/figure/Photomicrograph-ofDiff-Quik-stained-blood-smear-from-black-rhinocerosSiabuwa-infected_fig1_322072881

H&E Staining of Spleen Tissue in the PBS- treated Control Mice (A), PBStreated (B), rmIL- 18Fc Chimeratreated (C) and rmIL- 18-treated (D) Malaria Mice. Spleen of infected mice treated with PBS and rmIL-18 showed enlargement of red and white pulp elements accompanied by loss of typi- cal structure of germinal center leading to splenomegaly (B, C and D). Malarial mice treated with rmIL-18Fc chimera showed improved morpho- logical and histopathological changes (C) (H&E staining, 100x) https://www.researchgate.net/figure/H -E-Staining-of-Spleen-Tissue-in-the-PBStreated-Control-Mice-A-PBS-treatedB_fig3_284888721 High magnification H&E stains of lymph nodes from mice lacking IRF-1. Lymph nodes from Rb þ / À IRF þ / þ mice demonstrate a normal small lymphoid population. https://www.researchgate.net/figure/Hig h-magnification-H-E-stains-of-lymphnodes-from-mice-lacking-IRF-1-Lymphnodes-from-Rb_fig1_9886657

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Identifying Immune Cells and Structures Staining methods useful for diagnostic purpose, but distinguishing morphological differences can be difficult Use Antibody Visualisation

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Antibody Visualisation - determine cell surface phenotype of present cells cannot be seen by naked eye Agglutination can be macroscopically observed Can use antibodies to see binding sites

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Immunofluorescence and Immunohistochemistry Antibody bind to antigen = detect molecules in cell / tissue / fluid Stable antibody-antigen binding, can wash unbound antibodies Visualisation: fluorescent dye/enzyme > colour > detected using microscope or spectrophotometer Comparing Immunofluorescence Methods

Immu2011 Practical Summary notes

Direct Immunofluorescence Method

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Advantages

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Disadvantages •

Indirect Immunofluorescence

Combined

Label antibody with • fluorescent dye eg fluorescein / rhodamine UV light exposure > excite fluorochrome Characteristic wavelength = colour = antibody location

Specific secondary antibody labels primary antibody bound to antigen

Rapid, single step • Use multiple antibodies from same • host

Secondary amplifies • signal Few labelled secondaries can detect many • primaries

Amplify signal for weaker targets with secondaries Stain with multiple primaries rom same species

No signal amplification from secondary antibody

Two step staining • Requires antibodies from diff hosts

Multi-step staining

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Immu2011 Practical Summary notes

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Examples

Each primary must be labeles individually Step 1: CF 488A mouse anti-A +CTF568 mouse antiB

Step 1: Mouse anti-A + Rabbit anti-B Step 2: CF 488a goat anti mouse + CF 568 goat anti-rabbit

Step 1: Mouse anti-A + Rabbit anti-B Step 2: CF 488a goat anti mouse + CF 568 goat anti-rabbit Step 3: CF640R mouse anti-C

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Immunohistochemistry Use of enzyme labelled antibodies Reveal corresponding antigens Colourless substrate added to slide, binds to antigen > enzyme converts substrate to colour > observed using light microscope Prac 1 Activity 2 - identifying examples of immunohistology / samples

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Identify if direct/ indirect immunofluorescence Identifying cell types in sheets eg dendritic, and immunological function of cells Prac 2 - Immunology Lab Techniques Behave appropriately in an immunology laboratory • Correct footwear - closed ,impermeable • Safety glasses and lab coats • Gloves and hand washing • No eating / drinking • Perimeter equipment: biosafety cabinet, fumehood, centrifuge/PCR machine/plate-reader • Waste disposal • Alarms and evacuation procedure Use a pipette properly Pipettes Accurate Use • Small volumes of expensive reagents eg antibodies • Time consuming to make • Technique = less error = more accurate results Range of Pipettes - 4 main sizes • P10 (pink) : 1-10uL • P20 (turquoise) : 2-20uL • P200 (yellow) : 20-200uL • P1000 (blue) : 100-1000uL.

Immu2011 Practical Summary notes

P1000 pipette tips

P200 pipette tips

P20 and P10 pipette tips

Scientific Notations and Small Volumes Litre Millilitre Microlitre Nanolitre

1/1 of a litre 1/1000 of a litre 1/1,000,000 of a litre 1/1,000,000,000 of a litre

100

L

-3

mL

-6

uL

-9

nL

10 10 10

Immu2011 Practical Summary notes Picolitre

1/1,000,000,000,000 of a litre

10-12

pL

How to use a pipette • • • • • • • •

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Attach appropriate pipette tip to pipette Dial required volume (uL) Hold pipette to first stop Submerge tip below surface and slowly release pressure from stop Check for air bubble / gaps = accuracy Place tib in receiving tube, push button to second stop Use same tip for same solution Eject into sharps bin

Accuracy of pipette Using: coloured deionised water, precision balance, waste container, pipette to test, appropriate tips for the pipette Protocol •

Place the waste container onto the balance and tare the balance.

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Set the volume on the pipette to the maximum volume the pipette can deliver and attach a tip to the pipette. Take up deionised water into the tip. Dispense the water into the waste container on the balance. Record the weight. Tare the balance and repeat steps 3-4 three more times. Discard the tip.

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1. Set the volume on the pipette to the middle of the volume range for the pipette and attach a tip to the pipette. 2. Repeat steps 3-5. 3. Set the volume on the pipette to the minimum volume the pipette can deliver and attach a tip to the pipette. 4. Repeat steps 3-5. 5. For each volume setting, calculate the average weight of the volume delivered. Use these values and the equation below to determine the mean error, and therefore the accuracy of your pipette.

NOTE: The acceptable error for pipettes will depend on the manufacturer, but mean error greater than 1% indicates inaccurate pipette needs calibrating and contact manufacturers

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Prac 2 Student Activity 1 - pipette identify suitable tip for pipette determine accurate pipettes Understand how to make dilutions Perform simple calculations required in an immunology laboratory Dilutions and Calculations

Immu2011 Practical Summary notes •

Practical format: x:y ration used

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Eg 1mL of 1:20 dilution is 50 uL of x to 950uL of y 1mL / 20 = 50uL , then 1mL - 50uL = 950uL

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Prac 1 Student Activity 2 - dilution and calcs Determine dilution factor required to dilute a solution Volume of dilute and volume of cells to suspend Pipettes required for dilutions NOTE: A 1% w/v solution is defined as 1g dissolved in 100mL.

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Understand how to use a haemocytometer to count viable cells accurately Cell Counting Convention for counting cells: Cells falling across the top and left border lines of the square are considered to be in the square, whereas cells on the bottom and right borders are excluded.

Concentration of cells (cells/mL in original suspension:

Sources of Error Haemocytometer counts are subject to the following sources of error: •

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It is assumed that the total volume in the chamber represents a random sample. This will not be a valid assumption unless the suspension consists of individual separated cells. Unless 90% or more of the cells are free from contact with other cells, the count should be repeated with a new sample. A sample will not be representative if the cells are permitted to settle before a sample is taken. Always mix the cell suspension thoroughly before sampling. Unequal cell distribution in the sample. Improper filling of chambers/flood into adjoining channels. Failure to adopt convention for counting cells in contact with boundary lines or with each other. Statistical error.

Prac 3 - Antigen-Antibody (Ag-Ab) Interactions : Agglutination

Immu2011 Practical Summary notes

Learning Outcomes At the conclusion of this practical you should be able to: •

To understand what polyclonal antibodies are and where we get them from for our research. Immunisation • Develop antibodies during pathogen exposure : infection or immunisation • Deliberate exposure = generate antibodies • Methods of administration: intravenous oral, subcutaneous, intradermal, and intraperitoneal injections or the antigen can be painted on the skin (cutaneous). Generates polyclonal antibody response: mixture of antibodies produces by b • cell clones recognising different epitopes on same antigen (one b cell produces one specific antibody type) •

Experimental example: rabbit injected with sheep red blood cells (SRBC) > generate anti-SRBC antibodies • After 3-4 weeks : same SRBC antigen administers • After another 3 weeks: blood taken from rabbit > agglutinate > serum containing anti-SRBC antibodies obtained Agglutination - used to observe Ag-Ab interactions • Result from 3D lattice of antibody-epitope binding (antibodies have at least 2 antigen binding sites eg IgG has 2) • Foreign RBC combine with specific antibody > form clumps • Specific antibody reacting with RBC in optimal proportions = haemagglutination (agglutination of cells) IgM 10 is most efficient (10 binding sites) • Presence of specific antibody in serum: diagnostic of active / past infection • Need to be quantified by reading estimate of titre of amount of present antibodies Titre: reciprocal of the highest dilution of antibody that causes a detectable • reaction (e.g. dilution 1:320 = titre 320/1 = 320). Other uses of agglutination for Ag-Ab interaction: • RBC coated with OTHER antigens as indicator cells in agglutination reactions with appropriate antisera • Coated cells can be replaced by coated latex beads (eg RapiTex for detecting rheumatoid factor in serum in prac part 2) • Diagnosis, microorganism identification, type blood for transfusion, tissue for transplant •

How to perform serial dilutions properly.

Immu2011 Practical Summary notes How to use the principles of antibody binding in a visual immunology assay. • To “see” cross-reactivity in action. Haemagglutination Assay •

Protocol • Method summary: titrate polyclonal antibodies via agglutination to find antiSRBC antibodies, anti-SRBCE reacted with horse RBC (HRBC) ie cross-reactivity Reagents • • • • •

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Phosphate buffered saline (labelled 'PBS') Sheep red blood cells (labelled 'SRBC') in suspension in PBS (2% v/v) Horse red blood cells (labelled 'SRBC') in suspension in PBS (2% v/v) Serum sample containing anti-SRBC antibodies (labelled 'anti-SRBC') Normal rabbit serum (labelled 'NRS')

Equipment • • • • • •

96 well V-bottom microtitre plate P200 pipette P200 multichannel pipette P200 tips Reagent well 37°C incubator

Method Prepare two-fold serial dilutions of each serum sample in the wells of the microtitre tray starting at 1:2 dilution as described below: Place 50μl PBS in all wells of rows A, B C and D. Add 50µl serum containing antibody (anti-SRBC antibodies ie. serum from rabbit immunised against SRBC,) to well 1, rows A and C. Add 50µl normal serum (from an unimmunised rabbit) to well 1, rows B and D. In each row, mix serum and PBS by pipetting up & down 3-4 times, then transfer 50µl of this 1:2 dilution of serum to well 2. Discard the pipette tips. Put fresh tips onto your pipette, then mix (as above), then transfer 50µl of this 1:4 dilution of serum to well 3. Continue to well 12. Discard 50μl from this well. Add 50µl of SRBC suspension to all wells in rows A and B. Add 50ul HRBC suspension to all wells in rows C and D. Label the plate with your name. Incubate 1...