WEEK 3 Attachment Sharing

Hello guys, it's my turn to share my SIP experience with you! =)

Anyway, for your information, i am currently working at a Oncology (cancer) research institute and everyone in my group is actually pretty much working on their own projects though we have general lab meetings every alternate weeks to update each other on our research progress etc etc.

As for me, i am focusing on a particular gene (let's just call it gene A) to see if there is an over-expression of it in prostate cancer. If there is a huge amount of gene A found in the prostate cancer cells, we can then make assumptions that perhaps this gene A has something to do with encouraging tumour angiogenesis or cancer cell proliferation etc etc. From it, we will then be able to find out if we could come up with an inhibitor that can block the action of the proteins (translated from gene A) and hopefully treat this cancer! (ok, this is the simplified version of my job scope. it's so much more complicated if i really want to elaborate but i will just leave it as that ok? =] )

For the past 3 weeks, i have been trained on Immunohistochemistry (IHC) which is very commonly used in cancer research to allow one to check if a particular protein is present/over-expressed in the cancer cells of that particular organ of the body. If you can still recall what we learnt during HistTech, it's 90% exactly the same as the staining we did =)

IHC: Materials and Methodology

1) As tissue samples from hospitals normally are formalin-fixed/paraffin-embedded sections, deparaffinization was done in 3 consecuetive xylene baths for 5 mins each

2) The slide was then dipped into 2 changes of absolute alcohol for 5 mins each as well.

Significance: To remove xylene from the slides and to prepare the sections for rehydration. This is because xylene and water are NOT miscible.

3) Rehydration was then performed as the slide was dipped in 95% alcohol (2 changes, 5mins each) before it was placed in 75% alcohol (2 changes, 5mins each). Rehydration is considered complete when the slide was rinsed with tapwater.

4) Pre-treatment was carried out as the slide was immersed in Ag Retrieval Buffer before it was placed in the Mega Microwave Histoprocessor at 117 degree Celsius for 30 mins.

Significance: When you fix a tissue sample using formalin, what it does is to denature proteins by coagulation, by forming additive compounds or by a combination of the 2. As a result, confomational changes in the protein structures will then occur to inactivate the enzymes and preserve the cells/tissues. Therefore, this pre-treatment process is to "revive" the immunoreactivity of the Ag to an immunogen. This process is aka "epitope retrieval" process.

5) The slide was cooled for 20mins before it was dripped dry and encircled using a Dako Pen

Significance: Dako pen is able to "trap" whatever reagents used on the sample within the encircled area so we don't have to use too much reagents and waste them.

6) Peroxidase block was added for 10 mins to the section to block any endogenous peroxidase that was produced by the cells.

Significance: Our secondary Ab will be labelled with Horseradish Peroxidase. Hence, we don't want any false positive results because of the presence of endogenous peroxidase.

7) Slide was washed to remove any excess peroxidase block reagent.

8) Ready-to-use Protein Block was used on the slide for 1 hour

Significance: Remember we have learnt about the "blocking" step in MBio last semester? It works in the same theory: to prevent non-specific binding.

DO NOT WASH SLIDE THIS TIME ROUND because even if you feel that the protein block might block the antigens in which the primary Ab have to bind to, this is not true. According to my senior, as the primary Ab used is more specific to the Ag than the protein block, it will have a higher affinity and the former can eventually replace the protein block. Hence, the end results will not be affected.

9) Primary Ab was then added to the slide for 1 hour. For every different kind of Ab used, we have a specific dilution for it. In case you are curious to know, i am using 1:5000 dilution =)

10) Washing was done this time to remove excess Ab

11) Secondary Ab labelled with HRP was added. Since my primary Ab is raised in mouse, my secondary Ab is an anti-mouse solution yup!

12) Washing carried out again

13) Chromogen and DAB was then added for just 5 minutes.

Significance: DAB will oxidise when it comes into contact with the air in the environment causing polymerisation and increasing its staining intensity. Chromogen is the substrate that will bind to the HRP to give us the signal we want.

14) After the slide was rinsed with tap water, it was counterstained using only Mayer's Haematoxylin.

Significance: As recent research has shown that protein A (from gene A) will be localised only in the nucleus, we only have to use haematoxylin and can save on our Eosin dye.

15) Dehydration was then carried out as the slide was dipped into 75% alcohol (2 changes, 5 mins each), 95% alcohol (2 changes, 5 mins each) and lastly absolute alcohol (2 changes, 5 mins each)

16) The slide was placed in the fume hood for 10 mins for drying to completely remove any water present on the slide/sample

Significance: water and xylene = NOT miscible

17) Lastly, the slide was placed in xylene for 3 changes for 5 mins each before it's mounted using DPX

Example:

Figure 1. Prostate cancer tumour sample expressing gene Z (Nucleus gives dark brown coloration). Note that only Mayer's Haematoxylin was used because researchers already know that gene Z staining is mainly localised in the nucleus.

Image is taken with permission from fellow co-worker.

Hope i didnt get my concepts wrong. LOL. correct me if you think i am inaccurate in any way yup =) Anyway, hope you guys are enjoying SIP over at your side.

Psst: should u have too much time to spare, u can join me for lunch coz i m alone like nisha as well! haha.

KangTing TG02

0503331A