TITLE: Staining Protocols for Safety Study of Wireless Fecobionics Device
AUTHORS: Yanmin  Wang, Ghassan Kassab, Hans Gregersen, Bhavesh Patel

[DESCRIPTION]
Protocols for Hematoxylin and Eosin staining and Immunofluorescence for two SPARC datasets: Safety Study of Wireless Fecobionics Device and Safety testing of predicate device for Fecobionics. The procedures were approved by Calmi2 Institutional Animal Care and Use Committee (IACUC)

Mongrel dogs were randomly assigned into two groups: the Fecobionics device group, and the THD SensyProbe control group. All animals were anesthetized, the devices were transanally inserted into the rectum, and then inflated at 20 ml. Six minutes later the devices were removed either naturally or manually. The animals in each group were randomly assigned into two subgroups, acute and chronic. The dogs in the acute subgroups were euthanized immediately after the removal of the devices, whereas the dogs in the chronic groups were euthanized two weeks after the device removal. The tissue samples were harvested upon sacrifice. 



[STEPS]
SECTION: Hematoxylin and Eosin Staining
1. Fix the tissues with 4% paraformaldehyde (pH 7.4) overnight at4 °C 

SECTION: Immunofluorescence
16. Put the frozen slides at room temperature for 30min.
SECTION: Hematoxylin and Eosin Staining
2. Embed the tissues with Optimal Cutting Temperature (OCT) compound and freeze with liquid nitrogen. 
SECTION: Hematoxylin and Eosin Staining
3. Cut 7 µm sections with cryostat (Leica, CM1850).
SECTION: Hematoxylin and Eosin Staining
4. Rinse slides in 1X PBS for 3 minutes.
SECTION: Hematoxylin and Eosin Staining
5. Rinse in a gentle stream of tap water until OCT is washed off for 3 minutes.
SECTION: Hematoxylin and Eosin Staining
6.  Stain in Hematoxylin for 20 minutes.
SECTION: Hematoxylin and Eosin Staining
7. Wash in a gentle stream of tap water for 1 minute, then dip in saturated lithium carbonate solution for 45 seconds.
SECTION: Hematoxylin and Eosin Staining
8. Blue nuclei in 1X PBS for 20 seconds.
SECTION: Hematoxylin and Eosin Staining
9. Wash in a gentle stream of tap water for 1 minute.
SECTION: Hematoxylin and Eosin Staining
10. Dip in 70% EtOH for 30 seconds.
SECTION: Hematoxylin and Eosin Staining
11. Dip in 95% EtOH for 30 seconds.
SECTION: Hematoxylin and Eosin Staining
12. Counterstain in Eosin Y for 10 seconds.
SECTION: Hematoxylin and Eosin Staining
13. Dehydrate through 2 changes of 95% EtOH for 15 seconds.
SECTION: Hematoxylin and Eosin Staining
14. Dehydrate through 3 changes of 100% EtOH for 15 seconds.
SECTION: Hematoxylin and Eosin Staining
15. Clear through 3 changes of Xylene for 1 minute each and coverslip.
SECTION: Immunofluorescence
18. 1% SDS for 5min, then wash 3 times.
SECTION: Immunofluorescence
17. Wash slides with PBS 3 times. Each time 5 min.
SECTION: Immunofluorescence
19. 0.25% triton in 1xPBS for 15min.
SECTION: Immunofluorescence
20. 10% Donkey serum in 1xPBS for 1hour.
SECTION: Immunofluorescence
21. Incubate slide with primary antibody diluted in 0.25% triton 2.5% Donkey serum 1xPBS at room temperature for 3 hours.
SECTION: Immunofluorescence
22. Wash slides with PBS 3 times. Each time 5 minutes.
SECTION: Immunofluorescence
23. Incubate slide with secondary antibody (1:100 dilution) for 1 hour.
SECTION: Immunofluorescence
26. Wash slides with PBS 2 times. Each time 5min.
SECTION: Immunofluorescence
28. Dry and mount coverslip.
SECTION: Immunofluorescence
24. Wash in PBS 3 times. Each time 5 minutes.
SECTION: Immunofluorescence
25. Stain with Wheat Germ Agglutinin (1:100 dilution) for 30 minutes.
SECTION: Immunofluorescence
27. Stain with Hoechst 33442 (1:100 dilution) for 30 minutes.