TITLE: Immunohistochemistry using paraffin embedded tissue
AUTHORS: Michael J Hurley

[DESCRIPTION]
This protocol describes how to stain paraffin wax embedded sections of tissue for alpha-synuclein by immunofluorescence or DAB/peroxidase immunohistochemistry and is based on Hurley et al., 2013 (https://doi.org/10.1093/brain/awt134). 

The protocol works for many other antibodies and the only change necessary for most antibodies is determining the optimal dilution of the primary antibody and the best type of antigen-retrieval to use.

[STEPS]
SECTION: Wax embedding
1. Dissect tissue (fresh or trans-cardially perfused with formalin (10% neutral buffered formaldehyde) or 4% paraformaldehyde in PBS).

 


SECTION: Wax embedding
2. Place tissue in at least 5 volumes of fixative at 4 °C for > 2880 min if tissue was perfused or > 5760 min 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.
SECTION: Wax embedding
3. 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.

 

SECTION: Immunofluorescence staining
24. Wash with PBS 3 x 5 min

SECTION: DAB/peroxidase staining
31. Incubate sections in biotinylated goat anti-rabbit IgG (1:250) secondary antibody for 2 hours.

SECTION: Immunofluorescence staining
26. Mount sections using Hydromount™ (National Diagnostics). 
SECTION: DAB/peroxidase staining
33. Incubate sections with streptavidin-peroxidase conjugate (1:250) for 1 hour.
SECTION: DAB/peroxidase staining
34. Wash PBS 4 x 5 min

SECTION: DAB/peroxidase staining
35. 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 3 min,

SECTION: DAB/peroxidase staining
37. Counter stain with haematoxylin 5 s to 10 s if desired.

SECTION: DAB/peroxidase staining
38. Wash sections with running tap water until it runs clear to remove excess stain.
SECTION: Wax embedding
4. Mount the tissue in a block of wax using an embedding station and metal or plastic moulds.  
Store at Room temperature

SECTION: Wax embedding
5. Section the tissue (  to  )using a microtome. 
Store sections at Room temperature

SECTION: Dewaxing and antigen retrieval
8.  Dewax sections in two changes of Histo-Clear II (National Diagnostics).
1.  10 min
2. 10 min

SECTION: Dewaxing and antigen retrieval
9. Rehydrate sections through graded (v/v) ethanol solutions.
1.       100 %    5 min
2.       100 %    5 min
3.        95 %    5 min 
4.        80 %    5 min
5.        70 %     5 min
SECTION: Dewaxing and antigen retrieval
10. Wash with PBS 5 min

SECTION: Dewaxing and antigen retrieval
12. Wash with PBS 5 min

SECTION: Dewaxing and antigen retrieval
13. For antigen retrieval heat 100 °C  the sections in 15 mM sodium citrate buffer containing 0.1% Tween® 20, pH 6 for 25 min in an 800-watt microwave set on 30 % power.
SECTION: Dewaxing and antigen retrieval
14. Cool with running tap water 5 min

SECTION: Immunofluorescence staining
17. Draw around the sections with a wax pen. 


SECTION: Immunofluorescence staining
19. 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) 1200 min    Room temperature 

SECTION: Immunofluorescence staining
20. Wash with PBS 4 x 5 min

SECTION: Immunofluorescence staining
22. Wash with PBS 2 x 5 min
SECTION: Dewaxing and antigen retrieval
7. Heat sections 60 °C 30 min
SECTION: Dewaxing and antigen retrieval
11. Quench endogenous peroxidase with 0.3 % hydrogen peroxide (v/v) 5 min

SECTION: Dewaxing and antigen retrieval
15. Wash with PBS 2 x 5 min

SECTION: Immunofluorescence staining
21. incubated sections with Alexa Fluor goat anti-rabbit IgG (1:200) (A27034) 90 min 
SECTION: Immunofluorescence staining
25. Dip slides in water to remove salts 
SECTION: Wax embedding
3.1. Gut tissue
1.      10 % formalin              1 min  Room temperature
2.     10 % formalin              1 min  Room temperature 
3.     70 % ethanol               60 min  30 °C 
4.     90 % ethanol               60 min  30 °C 
5.     100 % ethanol             60 min  30 °C 
6.     100 % ethanol            60 min  30 °C
7.     100 % ethanol             60 min  30 °C
8.     100 % ethanol             80 min  30 °C 
9.     xylene                          60 min  30 °C 
10.    xylene                         60 min  30 °C 
11.     xylene                         80 min  30 °C 
12.    paraffin wax               80 min  62 °C 
13.    paraffin wax               80 min  62 °C 
14.    paraffin wax               80 min  62 °C 

SECTION: Wax embedding
3.2. Brain tissue (whole mouse)
1.     10 % formalin              1 min  Room temperature
2.     10% formalin              1 min  Room temperature 
3.     70% ethanol              120 min  30 °C 
4.     90% ethanol              120 min  30 °C 
5.     100% ethanol             120 min  30 °C 
6.     100% ethanol            120 min  30 °C
7.     100% ethanol             120 min  30 °C
8.     100% ethanol            120 min  30 °C 
9.     xylene                        120 min  30 °C 
10.    xylene                       120 min  30 °C 
11.    xylene                        240 min  30 °C 
12.    paraffin wax             120 min  62 °C 
13.    paraffin wax             120 min  62 °C 
14.    paraffin wax             120 min  62 °C 

SECTION: Dewaxing and antigen retrieval
6. Select slides to be stained and place in a microscope slide rack. 
SECTION: Immunofluorescence staining
23. incubate sections with DAPI (1 µg/ml)  5 min

SECTION: Dewaxing and antigen retrieval
16. For immunofluorescence staining go to step 17 

For DAB/peroxidase staining go to step 27
SECTION: Immunofluorescence staining
18. Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 90 min
Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.
SECTION: DAB/peroxidase staining
37.1. 
SECTION: DAB/peroxidase staining
40. Clear sections with Histo-Clear II (National Diagnostics) 2 x 5 min

SECTION: DAB/peroxidase staining
39. Dehydrate sections through graded (v/v) ethanol solutions
    70 %       5 min
    80  %      5 min
    95 %       5 min
    100 %     5 min
    100  %    5 min

SECTION: DAB/peroxidase staining
41.  Mount with Omnimount™ (National Diagnostics) or equivalent permanent resin.
SECTION: DAB/peroxidase staining
36.  Wash sections for > 5 min in running tap water. 

SECTION: DAB/peroxidase staining
32. Wash PBS 4 x 5 min
SECTION: DAB/peroxidase staining
29. 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) 1200 min    Room temperature 
SECTION: DAB/peroxidase staining
30. Wash PBS 4 x 5 min
SECTION: DAB/peroxidase staining
27. Draw around the sections with a wax pen. 
SECTION: DAB/peroxidase staining
28. Block non-specific binding sites with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal 90 min
Depending on the size and number of sections 150-250 microliters of solution should be sufficient to cover the sections.