2nd day at IMCB
Ok.. this is a bit late but I shall now blog day 2 at IMCB. Ok, basically, that day, I went in prepared to do alot of waiting (which is why my laptop got the chance to sit on a genuine IMCB desk that day). We went up to the lab and put on lab coats to begin. Basicallly, that day was supposed to be the time where the real stuff happens. The first day was just getting the zebra fish embryos used to non MEOH solutions to prevent shock. Today, we finally begin teh real stuff. Primary antibody solution applied in day 1 was removed and PBDT was re added to wash off the primary antibody staining to ensure that background staining is kept to a minimal. This consisted of three times replacing of the PBDT at half hour intervals. Total slack time: 1.5 hrs!! After that, add secondary antibody solution and incubate for 2hrs. The secondary antibody solution binds to the primary antibody for the binding of the stain to the secondary antibody later on. Total lunch time: 2hrs!! For those interested in what we did for lunch, we just went to teh food court next door. Reasonable tasting food :)
Ok. After the 2hrs of lunch came another wash cycle!! Same as before, replacing the PBDT at half an hour intervals 3 times was our job. Total slack time: 1.5hrs!! After which, an AB complex is added which binds to the biotin in the secondary antibody solution and that allows teh stain to bind to the AB complex, which is bound to the secondary antibody, which is bound to the primary antibody, which is bound to the myosin.... yar.... long way to go.. After adding the AB complex, the eppendorf tubes are agintated at low speed for 45 mins. Slack time: 45mins!!!
After that, the famous wash cycle again!!! This time though, they decided to cut slack time. So this time, the was cycle was set to replace solution at 15mins intervals 4 times. This time though, the solution being replaced is Phosphate Buffer (PB) instead of PBDT. Yar.. so total slack time: 1hr!!!
After all that slacking and waiting, we finally got to another crucial step: Staining!! Using staining solution apparently able to cause cancer, the embryos are introduced to staining solution and placed under a microscope where 5 microlitres of H2O2 or hydrogen peroxide is added to initiate staining. Once the embryos look stained enough, teh embryos are flooded with PBS to stop the staining. This is to ensure that the whole embryo does not become stained or rather to minimize background staining. After this, 80% glycerol is added to cause the embryo's to be as translucent as possible for viewing under microscope and the day finally came to an end. Oh yeah, forgot to tell you, from teh staining part onward, it is all done on a watch glass instead of the eppendorf tube. Easier to see, especially under microscope.
Yar. Basically, today's slack time came to about 5 hrs of slack time (1.5 + 1.5 + 1 + 45mins) excluding lunch by the way. So yes, its more slack than school... :P
We had fun i think. Today, everyone seems to be more friendly instead of totally diao like the first day. :) cheers for us!!!!
So ends the second day.....
Ok. After the 2hrs of lunch came another wash cycle!! Same as before, replacing the PBDT at half an hour intervals 3 times was our job. Total slack time: 1.5hrs!! After which, an AB complex is added which binds to the biotin in the secondary antibody solution and that allows teh stain to bind to the AB complex, which is bound to the secondary antibody, which is bound to the primary antibody, which is bound to the myosin.... yar.... long way to go.. After adding the AB complex, the eppendorf tubes are agintated at low speed for 45 mins. Slack time: 45mins!!!
After that, the famous wash cycle again!!! This time though, they decided to cut slack time. So this time, the was cycle was set to replace solution at 15mins intervals 4 times. This time though, the solution being replaced is Phosphate Buffer (PB) instead of PBDT. Yar.. so total slack time: 1hr!!!
After all that slacking and waiting, we finally got to another crucial step: Staining!! Using staining solution apparently able to cause cancer, the embryos are introduced to staining solution and placed under a microscope where 5 microlitres of H2O2 or hydrogen peroxide is added to initiate staining. Once the embryos look stained enough, teh embryos are flooded with PBS to stop the staining. This is to ensure that the whole embryo does not become stained or rather to minimize background staining. After this, 80% glycerol is added to cause the embryo's to be as translucent as possible for viewing under microscope and the day finally came to an end. Oh yeah, forgot to tell you, from teh staining part onward, it is all done on a watch glass instead of the eppendorf tube. Easier to see, especially under microscope.
Yar. Basically, today's slack time came to about 5 hrs of slack time (1.5 + 1.5 + 1 + 45mins) excluding lunch by the way. So yes, its more slack than school... :P
We had fun i think. Today, everyone seems to be more friendly instead of totally diao like the first day. :) cheers for us!!!!
So ends the second day.....
