AIMS:            i)      To learn how to perform immunohistochemistry staining on paraffin-embedded sections using the indirect method and peroxidase- conjugated antibodies

ii)      To recognise the distribution of Glial Fibrillary Acidic Protein (GFAP) in sections of adult mouse brain

Safety warnings:

Wear gloves and an 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 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.

You will analyse your staining results in the next round of practicals – these two sessions together need to be written up as a lab report which is the coursework assessment on 4BBY1030 that counts for 30% of the final module mark.

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 Tris-buffered 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 the details of the constituents.)

in tubes:

in Coplin jars:

1. Tris-Buffered Saline (TBS)

2. Scott’s tap water

3. 70% alcohol (ethanol)

4. 90% alcohol (ethanol)

5. Absolute alcohol (100% ethanol)

6. Histoclear

other:

1. Beaker of tap water

2. Small bottle of Gill’s haematoxylin

3. 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  the entire section within the water-resistant ring with the rabbit anti-GFAP antibody. Leave for 30 minutes.

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 three or four sheets of tissue. 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.

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 analyse your slide under the microscope (next practical).