TITLE: Immunofluorescence staining  for tissue and organoids 
AUTHORS: Gabriela Vallejo Flores, Annika Fendler

[DESCRIPTION]
Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope.

This protocol will be used to analyze  the presence of some protein in Bladder tissue and organoids  tumor and non-tumor, some of the antigens are the following  CK20, CK14, CK5, CTK18. 

 

[BEFORE_START]

Before starting the protocol, place the primary antibodies on ice. 
Label the tubes for the primary antibodies dilutions 
During the incubation time of the blocking buffer, dilute the primary antibodies in blocking buffer B
Prepare the humidifying box 
use the pan pen to delimit the sample 

[GUIDELINES]
Prepare the solutions needed for this protocol one or two days before starting the sample processing: 
TBS 1x
TBS 1x 0.05% of Tween
Buffer A:  TBS  with 1% of BSA + 2% of host serum of  the secondary antibody
Buffer B:  with 1% of BSA 
Cuts the slides some days before the day of the staining 

[STEPS]
SECTION: Blocking
3. Add blocking buffer A (1% BSA + 2% of host serum (secondary antibody) in TBS 1x, add 150µl for tissue and 50µl for organoids staining, incubate the sample 60 min a room temperature in the humidifying box.
SECTION: Primary antibody
5. Add the primary antibody to the sample, dilute the antibody in blocking buffer B ( 1% BSA in TBS 1x) and Add 150µl for tissue and 50µl for organoids staining, incubate the sample a room temperature or  overnight at 4°C in a humidifying box. 


 

SECTION: Primary antibody
6. Remove the primary antibody with a towel in the corners around the sample.
SECTION: Primary antibody
7. Wash 3 times the sample with TBS 1X; 0.05% tween 20 and one time with TBS 1X.
   
SECTION: Secondary antibody
8. Add the secondary antibody to the sample, dilute the antibody in blocking buffer B, and add Hoesch 1:200 for nuclea staining, and incubate the sample 30 min a room temperature in a humidifying box. 


 

SECTION: Secondary antibody
9. Remove the secondary antibody with a towel in the corners around the sample.
SECTION: Sample Mounting
12. Add 2 drop of antifade solution( a mounting medium that Inhibits photobleaching of fluorescent dyes and fluorescent proteins, No warming necessary, Can be stored without sealing for long term analysis)
SECTION: Secondary antibody
10. Wash 4 times the samples with 0.05% tween in TBS 1x.

SECTION: Sample Mounting
13. Let dry the slides for 10 min a room temperature, then are ready for us.
SECTION: Sample Mounting
14. 
The slide can be keeping at 4°C for 1 or 2 weeks or minus 20 for some months. 
SECTION: Results
15. 
 

SECTION: Antibodies in use
1. 

 

SECTION: Blocking
4. Remove the blocking buffer A with a towel in the corners around the sample.
SECTION: Secondary antibody
11. Wash one time the samples with TBS 1x.
SECTION: Antibodies in use
2. Delimit the sample with a pan pen, add TBS 1x to the sample and check that the buffer is not licking.
SECTION: Results
16. 
 

SECTION: Results
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SECTION: Results
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SECTION: Results
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SECTION: Results
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