Histology and immunocytochemistry

Histology and immunocytochemistry

Sun et al., Sci. Transl. Med. 12, eaax2992 (2020)

1. Heart preparation

1). For paraffin sections, hearts were harvested and fixed in 10% neutral-buffered formalin for 48 h and cut into 2-mm-thick transverse slices using a matrix slicer (Zivic Instruments, Pittsburgh, PA) before standard paraffin embedding. Sections (5 µm) were thencut.

2). For cryosections, hearts were harvested and fixed in fresh ice cold 4% PFA for 24 h and then were dehydrated with gradient sucrose (10, 20, and 30% each for minimal 8 h) and then cut into 2-mm-thick transverse slices for optimal cutting temperature compound (OCT, Sakura) embedding before cryo-sectioning of 10-µm-thick sections.

2. Hematoxylin-eosin (Sigma-Aldrich)

1). Wash Paraffin sections in followingsolutions

1. Xylene (2 x 5 min)
2. 100% ethanol (2 x 5 min)
3. 95% ethanol (2 min)
4. 70% ethanol 2 x 2 min

2). Wash in PBS, 2 x 5 min

3). Rinse in dH₂O

4). Stain with filtered Hematoxylin (Sigma; MHS-16) for 6 min

5). Rinse in H₂O

6). Acid alcohol (~2 dips)

7). Rinse in H₂O

8). Rinse in 95% Alcohol for 1 min

9). Dip in Eosin 15-20 sec

10). 95% Alcohol briefly

11). 100% Alcohol 2 x 2 min

12). Dip in Xylene 2 x 2 min

13). Dry in fume hood

14). Permount

15). Store @ r.t

3. Picrosirius red (Abcam)

1). Wash Paraffin sections in following solutions

1. Xylene (2 x 5 min)
2. 100% ethanol (2 x 5 min)
3. 95% ethanol (2 min)
4. 70% ethanol 2 x 2 min

2). Wash in PBS, 2 x 5 min

3). Rinse in dH₂O

4). Stain with filtered Hematoxylin (Sigma; MHS-16) for 6 min

5). Rinse in H₂O

6). Stain in 0.1% picro-sirius red for one hour

7). Wash in two changes of acidified water (5 sec / change)

8). Dehydrate in three changes of 100% ethanol (5-10 sec / change)

9). Clear in 2 x 1 min of xylene 

10). Mount in perimount

11). Store @ r.t

4. Immunostaining

4.1 Paraffin Section Staining 

Regents

PBS

PBS containing Ca and Mg

Normal serum (species of secondary antibody)

Blocking buffer (1.5% normal serum of the species in PBS containing Ca and Mg) 
30% Hydrogen peroxide

Xylene

Ethyl alcohol, 100%, 95%, 70% 
ABC Elite (standard) Kit PK 6100 
Biotinylated secondary ImmPACT DAB

Permount for coverslip (Fisher SP15-100)

1. Wash Paraffin sections in following solutions

1. Xylene (2 x 5 min)
2. 100% ethanol (2 x 5 min)
3. 95% ethanol (2 min)

2. Quench endogenous peroxidase in cold methanol with 0.3-4% hydrogen peroxide, 30 min at r.t

3. 70% ethanol 2 x 2 min

4. Wash in PBS, 2 x 5 min

5. Antigen retrieval (0.01 M Citrate Buffer (pH 6.0) on hotplate, boiling for 20 min)

6. Cool the solution with slides in it to r.t (or at least for 10 min).

7. Rinse in PBS 2 x 5 min

8. Incubate in blocking buffer, 1 h,r.t.

9. Primary AB in blocking buffer O/N at 4°C

10. PBS, 3 x 5 min

11. Secondary Ab in blocking buffer, 1 h at r.t (Biotinylated goat anti rabbit, 1:350)

1. Meanwhile, prepare ABC
2. 2 drops of A to 5 ml of blocking buffer, mix
3. Add 2 drops of B, mix and let the complex form for 30 min (1 h better)

12. PBS, 3 x 5 min

13. Apply prepared ABC, 30-45 min, r.t., in moist chamber

14. PBS, 3 x 5 min

15. Equilibrate for 5 min in PBS (for DAB). Prepare DAB solutions (1 drop (~30 µl) DAB to 1 ml Diluent)

16. Apply DAB and monitor the time of development

17. Once developed, dip slides in water (~ 10 times)

18. PBS, 2 x 5 min

19. Counterstain

1. Rinse 2 x 5 min in dH₂O
2. Hematoxylin for 6 min (change for desired darkness)
3. Acid alcohol (~2 dips)
4. Rinse in dH₂O (repeat until water is clear, usually 2-3 times)

20. 70 % ethanol 1 min

21.100% ethanol (2 x 5 min)

22. Place in xylene, 2 x 5 min

23. Mount in Permount

Note:

1. Ku80 (1:400; Cell Signaling Technology), the sarcomeric myosin heavy chainCD31/PECAM (1:200; Novus), and GFP (1:1000; Novus), cardiac myosin heavy chain (1:3, Developmental Studies Hybridoma Bank)

1. For immunofluorescence-based stains, Use Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies or streptavidin (Thermo Fisher Scientific). No dehydration. Mount in Vectashield,

2. For bright-field detection, biotinylated goat anti-mouse or rabbit secondary antibodies (Vector Laboratories) were used in conjunction with the VECTASTAIN Avidin/Biotin Complex (ABC) Kit (Vector Laboratories) followed by alkaline phosphatase (Vector Red) or horseradish peroxidase/3,3′-diaminobenzidine (Vector Laboratories).
 

4.2 Cryosection Staining

2. Equilibrate slides to r.t

3. Wash 1 x 5 min with 1 X PBS.

4. Wash in PBT (PBS + 0.2 % Triton X-100), 3 x 2 min

5. Primary Ab: dilute in 1 X PBT, in the dark, wet chamber, r.t., 2 h or O/N at 4°C

6. Wash 1 X 5 min with 1 X PBS.

7. Secondary AB: dilute in 1 X PBT, in the dark, wet chamber, r.t., 1 h

8. Wash 2 X 5 min with 1 X PBS

9. To label nucleus, stain with Hoechst 33342 in PBS (1:1000, stock 1mg/ml), 5 min

10. Wash with PBS, 3 x 5 min

11. Mount in Vectashield, cover slip

12. Microscopy

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