Practical 3 - Immunohistochemistry PDF

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Cell Biology and Neuroscience – Practical 3 (Lap Report) notes  

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Immunohistochemical staining on mouse brain tissue to identify specific intermediate filament protein that marks glial cells Immunohistochemical staining use same framework as other stains b= but also use antibodies that are able to specifically recognise proteins and tissues to highlight specific cells/tissues Aim of staining is to identify a protein called Glial Fibrillary Acidic Protein (GFAP) Use images on KEATS to identify areas of mouse brain and look at distribution of GFAP and in which cells it is found GFAP highlight astrocytes

Types of glial cells    

Astrocytes mostly perform support functions – they regulate environment of neurons Oligodendrocytes – wrap around axon and provide insulation and myelination Ependymal cells – secrete cerebrospinal fluid and line ventricles Microglia – relatively small and perform immune function and allow phagocytosis

Introduction  Immunohistochemistry (IHC) combines histological, immunological, and biochemical techniques for the identification of individual tissue components by means of a specific antigen/antibody reaction tagged with a visible label  ‘histo’ = tissue  IHC makes it possible to visualize the presence and localization of specific cellular components within a tissue  Immunocytochemistry (ICC) refers to immuno-reactions on individual cells, usually extricated from surrounding tissue, or possibly grown in culture Direct method: primary antibody only  Underlying idea of immunohistochemistry is that we have a tissue (composed of cells) and cells within express (produce) a very specific tissue of interest  We then generate a primary antibody directed against antigen – in our case GFAP  We can link this antibody using a biochemical reaction to an enzyme called ‘Horseradish peroxidase (HRP)’.  If we now treat a tissue which expresses out antigen with an antibody linked to HRP, we end up with an enzyme HRP being concentrated in tissues that express antigen of interest  In summary, if a tissue is treated with HRP-antibody complex, HRP will be concentrated in areas where corresponding antigen is.  This is called direct method as primary antibody is attached to enzyme Indirect method: primary and secondary antibodies  We use a tissue that expresses antigen of interest – here it’s GFAP  A primary antibody will be made that detects this antigen  We inject this into tissue  We then use a secondary antibody labelled with HRP  This method is advantageous because each secondary antibody can bind to the heavy chain of the primary antibody creating multiple secondary antibodies bind to primary causing more HRP into the tissue  Allows more flexibility however this method can be expensive but more sustainable Detection of HRP 1

Cell Biology and Neuroscience – Practical 3 (Lap Report) notes      

We bind our primary antibody to antigen of interest We bind our secondary antibody to primary antibody, conjugated with HRP HRP is an oxidase meaning it can oxidase substances using H202 (hydrogen peroxide) Exogenous H202 can be added to stain as well as DAB (colourless) which when the two are oxidised by HRP, they form a brown DAB precipitate + H202 DAB is diaminobenzidine More DAB and more H202 we add, the more brown precipitate forms

Detection of GFAP  The purpose of P3 experiment is to look for GFAP which is expressed in mouse brain tissue  We have an antibody against GFAP which looks for GFAP specifically  GFAP is an intermediate filament so more likely to be inside cell – inside cytosol  There will be a secondary antibody  We will use H202 and DAB to form brown precipitate to show where GFAP is in the tissue  Primary antibody we are using is rabbit anti-GFAP antibody  Secondary antibody we are using is HRP-conjugated swine anti-rabbit IgG antibody Using a fluorescently labelled secondary antibody  Instead of using DAB and H202, we could have used a fluorescent dye such as: o Red fluorescence, blue nuclear counterstain (DAPI) o Mercury vapour UV light bulb (confocal microscopy)  Today, we will use haematoxylin to counterstain to get blue-purplish stain Schematic view of mid-sagittal section of mouse brain

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Olfactory bulb – much larger than others because mice have to smell a lot and rely on this Cerebral cortex – not folded, its smooth. Mice cortex is much smaller so does not require folding Corpus callosum – white matter which connects 2 hemispheres Hippocampus – centre for spatial activity Cerebellum – has folds

What the stained version of the mid-sagittal section looks like 2

Cell Biology and Neuroscience – Practical 3 (Lap Report) notes    

GFAP staining v Haematoxylin/eosin The latter show areas GFAP has been counterstained with Haematoxylin Areas in brown show where GFAP is located – corpus callosum and brown line going around

Staining in hippocampus Olfactory bulb

Cerebral cortex

Hippocampus

Corpus callosum

Dentatae gyrus

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Looking to the picture on the right, you can see blue counterstain as well as small brown dots which are cells that are positive for GFAP – these cells are astrocytes Astrocytes come in different sizes and shapes – some are quite fat e.g., protoplasmic astrocytes whilst spindley ones which are fibrous astrocytes Astrocytes tend to outline vessels – we can see that they outline vessels in brain which we know they do to allow access into blood brain barrier

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Cell Biology and Neuroscience – Practical 3 (Lap Report) notes Staining at the edge of cerebral cortex

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Edge of brain has GFAP on it too This shows cerebral cortex of mouse Haematoxylin counterstain shows arachnoid matter which is the blue stain between the ‘V’ shape You can see glial cells on surface on brain that are densely concentrated on the edge of the ‘V’

Why is GFAP important?  It is an intermediate filament (approx. 10 nm diameter) found in the cytoplasm (cytoskeleton) of astrocytes and ependymal cells. It is essential for normal astrocyte function and therefore it is required for: o Structural and mechanical support of the neuron o Communication between cells (both astrocyte-astrocyte and astrocyte-neuron) – includes signalling and transport communication o Integrity of the blood brain barrier – BBB protects against pathogens from entering brain GFAP is ‘essential’ – how do we know?  Loss of function experiment shows us why something is important or ‘essential’  Scientists have removed GFAP from mouse to see what the effect is without  Scientist found in normal mouse (left) there’s astrocytes v on the no GFAP mouse on the right’  Mice without GFAP show: o Abnormal myelination o Structural abnormalities in white matter o Blood brain barrier is functionally and structurally impaired

GFAP functions in injury and disease 4

Cell Biology and Neuroscience – Practical 3 (Lap Report) notes

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Scarring –is associated with prevention of nerve regeneration Astrocytoma – a brain tumour involving astrocytes – GFAP can help distinguish astrocytoma’s from other glial cells Alexander disease – GFAP can mutation can cause this disease causing abnormal IF proteins disrupting demyelination

Antibodies in research and medicine

Practical 3 - Immunohistochemistry AIMS:

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To learn how to perform immunohistochemistry on paraffin-embedded sections using indirect, peroxidase-conjugated antibodies To recognise the distribution of Glial fibrillary acidic protein in sections of adult mouse brain

Safety warnings: TBS, Scott’s water, 70% alcohol: 90%/100% alcohol, Histoclear: DAB:

IRRITANT FLAMMABLE TOXIC

Wear gloves and apron at all times. Confine waste reagents to disposable staining tray.

Overview: In this practical you will be conducting an immunohistochemistry (IHC) experiment to localize and visualize a protein in sections of mouse brain. Although several modifications of this procedure exist, it is very widely used in cell biology. The protein you will be detecting is Glial Fibrillary Acidic Protein (GFAP). This is an intermediate filament protein that can be used to distinguish astrocytes from other neuroglial cells. As laboratory practicals in the General Classroom are unfortunately impossible this year, you will perform the procedure in a lab simulation on KEATS. Representative results are also provided on

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Cell Biology and Neuroscience – Practical 3 (Lap Report) notes KEATS, and you will use those for your laboratory report which is the in-course assessment on this module. Materials: You have been provided with a slide containing a sagittal section of an adult mouse brain. It is a paraffin-embedded section that has already been dewaxed and rehydrated. It is provided in Trisbuffered saline (TBS) in a Coplin jar. Solutions: (Note that only partial information is given on the composition of the solutions. It is not necessary to know details of the constituents.) In tubes: 1. 2. 3. 4.

Blocking solution (2% bovine serum albumin in TBS) Primary antibody (rabbit anti-GFAP IgG, pre-diluted to 1:500 in TBS) Horseradish peroxidase (HRP)-conjugated secondary antibody (swine anti-rabbit IgG, conjugated to HRP and pre-diluted to 1:100 in TBS) Diaminobenzidine (DAB) solution (DAB diluted in Tris and including H2O2 as a substrate)

In Coplin jars: 1. 2. 3. 4. 5. 6. 7.

Tris-Buffered Saline (TBS) Beaker of tap water Scott’s tap water 70% Alcohol 90% Alcohol Absolute alcohol (100% ethanol) Histoclear

Other: 1. Small bottle of Gill’s Haematoxylin 2. Small bottle of DPX. Protocol: 1. Place your slide on the rack on the plastic tray. Using a disposable pipette, add sufficient Blocking solution to just cover the section entirely. Leave for 3 minutes. 2. Drain off the solution into the plastic tray. Using the same disposable pipette, cover entire section within the water-resistant ring with the rabbit anti-GFAP antibody. Leave for 30 minutes. 6

Cell Biology and Neuroscience – Practical 3 (Lap Report) notes

3. Drain off the solution into the plastic tray and place the slide in TBS (in the Coplin jar that you originally took the slide from) for 3 minutes. 4. Drain off the TBS into the plastic tray and replace the slide on the rack. Using a new disposable pipette, place just enough of the HRP-conjugated secondary antibody on the slide to cover the entire section. Leave for 30 minutes. 5. Wash again by returning the slide to the Coplin jar containing TBS for 5 minutes. 6. Drain off the TBS into the plastic tray and put the slide back on the rack. Using a new disposable pipette, cover the entire section with the DAB reagent within the water-resistant ring. Remember that DAB is toxic and so do this step CAREFULLY. Leave for 10 minutes. 7. Hold the slide with forceps and drain the DAB reagent by tapping the slide onto 3 or 4 sheets of tissues. Again, perform this step carefully and ensure that the small amount of waste DAB is confined to the tissues. Place the slide into the jar containing TBS. Leave for 1 minute. 8. Drain off the TBS into the plastic tray and put the slide back on the slide rack. Using a new disposable pipette add enough Gill’s haematoxylin for to cover the section. Leave for 30 seconds. 9. Drain off the haematoxylin into the plastic tray. Rinse the slide in the beaker of water to remove all of the haematoxylin. 10. Place the slide into the Coplin jar containing Scott’s tap water for 2 minutes. 11. Rinse the slide in beaker of water then wipe the back of the slide dry with a tissue, taking care not to touch the section on the other side. Place in the Coplin jar containing 70% alcohol. Lift slide up and down two times. Leave for 2 minutes. Dry your forceps. 12. Lift slide out, drain the excess alcohol into the jar, wipe the back of the slide, then place the slide in 90% alcohol. Leave for 2 minutes. 13. Lift slide out, drain the excess alcohol into the jar, wipe the back of the slide, then place the slide in 100% alcohol. Leave for 2 minutes. 7

Cell Biology and Neuroscience – Practical 3 (Lap Report) notes

14. Lift out the slide, then, holding it vertically, briefly drain by placing the end of the slide away from the label on to the surface of the blotting paper so that only the edge of the slide is touching and the absolute alcohol is draining down the slide onto the blotting paper. Then place the slide in the Coplin jar containing Histoclear and leave for 2 minutes. Agitate every 30 seconds by raising and lowering the slide in the Coplin jar two times. 15. Check your slide. If the section on it has a white emulsion/film, then go back to stage 14 and continue from there. 16. Apply a coverslip to the slide: Place a coverslip on a piece of blotting paper, then add one long ample streak of DPX mounting medium to the coverslip, using the wooden stick. Take the slide out of Histoclear, briefly drain but do not allow to dry out. Turn the slide around so the section is facing away from you. Lower the inverted slide onto the coverslip so the coverslip is covering the section. Press gently to ensure DPX spreads evenly under the coverslip. Lift up, wipe the back of the slide and make sure the coverslip is evenly placed. The DPX will polymerise to give a permanent preparation. You can now analyze your slide under the microscope. End of Practical 3

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