Practical 3 & 4: Immunohistochemistry Lab report
Introduction:
Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament found in astrocytes as well as many
other cells in the central nervous system. During this practical, we are aiming to visualise the distribution
of GFAPs in a sagittal brain section of an adult mouse, using indirect staining techniques. Indirect
staining includes two antibodies, where GFAPs will bind to primary antibodies (rabbit anti-GFAP
antibody), which is also bound to secondary antibodies labelled with horseradish peroxidase (HRP), on
paraffin embedded sections. HRP will catalyse the formation of a brown DAB precipitate when bound to
GFAP, allowing us to identify the location and abundance of GFAPs in the brain, as well as differentiate
them from other neuroglial cells under the light microscope. 119/150
Materials and methods:
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:
In tubes:
1. Blocking solution (2% bovine serum albumin in TBS)
2. Primary antibody (rabbit anti-GFAP IgG, pre-diluted to 1:500 in TBS)
3. Horseradish peroxidase (HRP)-conjugated secondary antibody (swine anti-rabbit IgG, conjugated
to HRP and pre-diluted to 1:100 in TBS)
4. Diaminobenzidine (DAB) solution (DAB diluted in Tris and including H2O2 as a substrate)
In Coplin jars:
1. Tris-Buffered Saline (TBS)
2. Beaker of tap water
3. Scott’s tap water
4. 70% Alcohol
5. 90% Alcohol
6. Absolute alcohol (100% ethanol)
7. 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.
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 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 onto 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.
Introduction:
Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament found in astrocytes as well as many
other cells in the central nervous system. During this practical, we are aiming to visualise the distribution
of GFAPs in a sagittal brain section of an adult mouse, using indirect staining techniques. Indirect
staining includes two antibodies, where GFAPs will bind to primary antibodies (rabbit anti-GFAP
antibody), which is also bound to secondary antibodies labelled with horseradish peroxidase (HRP), on
paraffin embedded sections. HRP will catalyse the formation of a brown DAB precipitate when bound to
GFAP, allowing us to identify the location and abundance of GFAPs in the brain, as well as differentiate
them from other neuroglial cells under the light microscope. 119/150
Materials and methods:
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:
In tubes:
1. Blocking solution (2% bovine serum albumin in TBS)
2. Primary antibody (rabbit anti-GFAP IgG, pre-diluted to 1:500 in TBS)
3. Horseradish peroxidase (HRP)-conjugated secondary antibody (swine anti-rabbit IgG, conjugated
to HRP and pre-diluted to 1:100 in TBS)
4. Diaminobenzidine (DAB) solution (DAB diluted in Tris and including H2O2 as a substrate)
In Coplin jars:
1. Tris-Buffered Saline (TBS)
2. Beaker of tap water
3. Scott’s tap water
4. 70% Alcohol
5. 90% Alcohol
6. Absolute alcohol (100% ethanol)
7. 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.
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 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 onto 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.
