RESEARCH ALBUM

Some of them are small fun projects, while some are serious ones.

Just as a photo captures the world differently from every angle, I hope my research could capture the world.

 
 
  • Minseok 'Joseph' Kim

Immunohistochemistry methodology - Optimal DAB Staining Time


With this result, I proved to change the protocol to optimize a procedure (immunohistochemistry) in the lab of Korean Medicine, Kyung Hee University.


Immunohistochemistry is a staining process that uses antibodies to visualize proteins (antigens) in tissue sections. It is a delicate process that requires multiple steps of washing and careful placement. Luckily I was able to learn this technique.


Here is the long protocol for the procedure:


Day 1

1. Rinse with 1X PBS (5 min. * 3)

2. H2O2 15 min

3. Repeat 1

4. Blocking for 1 hour in 0.3% BSA in PBST

5. Repeat 1

6. Attach primary antibody (c-fos = 1:1000 in PBST)

7. Overnight at RT


Day 2

1. Rinse with 1X PBS (5 min. * 3)

2. Attach secondary antibody (rb = 1:1000 in PBST), 1 hour at RT

3. Repeat 1

4. ABC (Avidin Biotin Complex) (4 drops of A, 4 drops of B per 10 ml in PBST)

5. Repeat 1

6. DAB (hydrogen peroxide 2 drops, DAB 4 drops, pH buffer 2 drops, NiCl 2 drops per 5 ml DW)

7. Attach to slide with coverglass


Towards the end of the protocol, there is a step where DAB (3,3′-Diaminobenzidine) is used as a chromogen to visualize the expressed protein.


In the lab, this duration of DAB staining was set to 30 sec ~ 1 min. During several tryouts in my learning period, however, I discovered the difference in time of the tissue submerged in this DAB solution resulted in different amount of visualized cells. This meant that if I'd put the tissues in the solutions longer, the results would change, causing inconsistency.


I became curious, and both keen, to correctly set standards for all of my slides. That's when I began my side experiment to determine the optimal time of DAB staining, at least for this particular experiment.


I first divided leftover tissues into groups and differed the staining time for each group.

As expected, more expressed cells were visible when staining time increased. To quantify, I counted these expressed cells from each group (n=20) and analyzed the results. The results were clear.


120-second staining showed optimal results. Too much staining or too light staining had caused cells to be visible/invisible, interfering with the experiment. The initial protocol of 30 sec ~ 1 min DAB staining was not to the optimal point, as 120 sec had better visibility and more expressed cell visible. Over 120 sec, the number of visible cells did not change much, but the high background made it harder to differentiate.


Thus, these results were used for future immunohistochemistry procedures in the lab. Other slides were all stained for 120 sec. Feeling proud I contributed to the lab.

Every photo on this page is taken by me, Minseok 'Joseph' Kim.