Immunohistochemistry using paraffin embedded tissue

Dissect tissue (fresh or trans-cardially perfused with formalin (10% neutral buffered formaldehyde) or 4% paraformaldehyde in PBS).

Place tissue in at least 5 volumes of fixative at 4°C for > 48h 0m 0s if tissue was perfused or > 96h 0m 0s if fresh. The tissue can stay in fixative indefinitely. If the tissue was fresh and the solution is turbid with blood, change the fixative after 24 hours.

Embed formalin fixed tissue in wax using an automated tissue processor. It can be done manually if you do not have access to an automated tissue processor. Histo-Clear™ II can be used instead of xylene.

Gut tissue

1.  10 % formalin              `0h 1m 0s`  `Room temperature`
   

2. 10 % formalin              `0h 1m 0s`  `Room temperature` 
   

3. 70 % ethanol               `1h 0m 0s`  `30°C` 
   

4. 90 % ethanol               `1h 0m 0s`  `30°C` 
   

5. 100 % ethanol             `1h 0m 0s`  `30°C` 
   

6. 100 % ethanol            `1h 0m 0s`  `30°C`
   

7. 100 % ethanol             `1h 0m 0s`  `30°C`
   

8. 100 % ethanol             `1h 20m 0s`  `30°C` 
   

9. xylene                          `1h 0m 0s`  `30°C` 
   

10. xylene 1h 0m 0s 30°C

11. xylene                         `1h 20m 0s`  `30°C` 
    

12. paraffin wax 1h 20m 0s 62°C

13. paraffin wax 1h 20m 0s 62°C

14. paraffin wax 1h 20m 0s 62°C

Brain tissue (whole mouse)

1. 10 % formalin              `0h 1m 0s`  `Room temperature`
   

2. 10% formalin              `0h 1m 0s`  `Room temperature` 
   

3. 70% ethanol              `2h 0m 0s`  `30°C` 
   

4. 90% ethanol              `2h 0m 0s`  `30°C` 
   

5. 100% ethanol             `2h 0m 0s`  `30°C` 
   

6. 100% ethanol            `2h 0m 0s`  `30°C`
   

7. 100% ethanol             `2h 0m 0s`  `30°C`
   

8. 100% ethanol            `2h 0m 0s`  `30°C` 
   

9. xylene                        `2h 0m 0s`  `30°C` 
   

10. xylene 2h 0m 0s 30°C

11. xylene 4h 0m 0s 30°C

12. paraffin wax 2h 0m 0s 62°C

13. paraffin wax 2h 0m 0s 62°C

14. paraffin wax 2h 0m 0s 62°C

Mount the tissue in a block of wax using an embedding station and metal or plastic moulds.

Store at Room temperature

Section the tissue (5 µm to 8 µm )using a microtome.

Store sections at Room temperature

Select slides to be stained and place in a microscope slide rack.

Heat sections 60°C 0h 30m 0s

Dewax sections in two changes of Histo-Clear II (National Diagnostics).

1. 0h 10m 0s

2. 0h 10m 0s

Rehydrate sections through graded (v/v) ethanol solutions.

1.   100 %    `0h 5m 0s`
   

2.   100 %    `0h 5m 0s`
   

3.    95 %    `0h 5m 0s` 
   

4.    80 %    `0h 5m 0s`
   

5.    70 %     `0h 5m 0s`
   

Wash with PBS 0h 5m 0s

Quench endogenous peroxidase with 0.3 % hydrogen peroxide (v/v) 0h 5m 0s

Wash with PBS 0h 5m 0s

For antigen retrieval heat 100°C the sections in 15 mM sodium citrate buffer containing 0.1% Tween® 20, pH 6 for 0h 25m 0s in an 800-watt microwave set on 30 % power.

Cool with running tap water 0h 5m 0s

Wash with PBS 2 x 0h 5m 0s

For immunofluorescence staining go to step 17

For DAB/peroxidase staining go to step 27

Draw around the sections with a wax pen.

Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 1h 30m 0s

Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.

Incubate with anti-alpha-synuclein antibody (1:500) (ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 20h 0m 0s Room temperature

Wash with PBS 4 x 0h 5m 0s

incubated sections with Alexa Fluor goat anti-rabbit IgG (1:200) (A27034) 1h 30m 0s

Wash with PBS 2 x 0h 5m 0s

incubate sections with DAPI (1 µg/ml) 0h 5m 0s

Wash with PBS 3 x 0h 5m 0s

Dip slides in water to remove salts

Mount sections using Hydromount™ (National Diagnostics).

Draw around the sections with a wax pen.

Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 1h 30m 0s

Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.

Incubate with anti-alpha-synuclein antibody (1:500) (ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 20h 0m 0s Room temperature

Wash PBS 4 x 0h 5m 0s

Incubate sections in biotinylated goat anti-rabbit IgG (1:250) secondary antibody for 2 hours.

Wash PBS 4 x 0h 5m 0s

Incubate sections with streptavidin-peroxidase conjugate (1:250) for 1 hour.

Wash PBS 4 x 0h 5m 0s

Visualise staining by incubation with PBS containing 0.05 % (w/v) 3,3′-Diaminobenzidine/0.015 % (v/v) H₂O₂/0.05 % (w/v) nickel ammonium sulphate for up to 0h 3m 0s,

Wash sections for > 0h 5m 0s in running tap water.

Counter stain with haematoxylin 0h 0m 5s to 0h 0m 10s if desired.

Wash sections with running tap water until it runs clear to remove excess stain.

Dehydrate sections through graded (v/v) ethanol solutions

70 %       `0h 5m 0s`



80  %      `0h 5m 0s`



95 %       `0h 5m 0s`



100 %     `0h 5m 0s`



100  %    `0h 5m 0s`

Clear sections with Histo-Clear II (National Diagnostics) 2 x 0h 5m 0s

Mount with Omnimount™ (National Diagnostics) or equivalent permanent resin.