WEEK 9

WOWWWW. Time flies. It's the 9th week =)) Hope u guys are coping well.

Anyway, this week, i will be talking about yet another skill that i picked up during my SIP - IMMUNOFLUORESCENCE. I believe just last week, Ye Tun has talked about this technique (http://sevenseven77.blogspot.com/) that he is using for viral identification. In my case, I am using it to show the localisation of a particular protein in the cell (whether it's in the nucleus or cytoplasm). It 's something really similar to Immunohistochemistry which i mentioned in WEEK 3 so i will skip explaining several steps ok? =)

Aim of test: I am using TWO primary Ab and of course 2 secondary Ab labelled with fluorescence substrate. The 2 fluorescence substrate i am using are FITC (giving the apple green fluorescence signal) and the TRITC (giving the red fluorescence signal) on one single section. Hence, when viewed under the fluorescence microscope, you should be able to see both colors!

Step 1 - 10 : Pls refer to the immunohistochemistry steps i wrote for WEEK 3

Step 11: Secondary Ab labelled with FITC was added. Since this 1st primary Ab that i am using is raised in mouse, my secondary Ab is an anti-mouse solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Step 12: Washing carried out

Step 13: This time round, the 2nd different kind of primary Ab was added

Step 14: Secondary Ab labelled with TRITC was added. Since this 2nd primary Ab that i am using is raised in rabbit, my secondary Ab is an anti-rabbit solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Significance: It is very important that you choose the primary Abs raised in different animals should you be using 2 antibodies on one single slide. This is because if both my 1st and 2nd primary Abs are raised in mouse, when secondary Ab is added, it will bind to BOTH the primary Abs instead of just one. Then, when you obtain your results, you wouldn't know if the Secondary Ab is binding specifically to its specific primary Ab.

Step 15: Washing was carried out once again.

Step 16: DAPI was added as a counter stain

Significance: DAPI is able to allow the nucleus of the cells to emit blue fluorescence signal. Hence, when viewed under the microscope, we would be able to know which cells are not emitting the fluoroscence at all (by only showing the blue DAPI fluorescence without the red/green fluorescence signal), hence showing no expression of that particular protein of interest.

Step 17: Washing done again

Step 18: Mounting medium was added onto the slide and cover-slipped.

Step 19: View under the fluorescence microscope! =)

As you can see from this pic, there are both the green and red fluorescence. In area where there is a slight tinge of yellow/orange, it is most likely where the 2 different proteins (Remember we are using 2 primary Abs?) co-localize. However, we need the confocal microscope in order to confirm if these 2 proteins are located in the exact same area.

Hope u guys understand my entry. haha. n take care =))

Chen Kangting
0503331A
TG02