Week 14- Confocal Microscopy!

Hey..this week’s my turn, and I have interesting PICS to share from my research work.

As I have mentioned before, my project is basically delivering DNAs into cancer cells using synthetic carriers such as polymers for future gene therapies. Hence, I’m doing the last part of my project which is ‘localisation of polymer-DNA complexes’ in the cells.

For the localization studies, my main aim is to determine whether my polymer goes into the nucleus. Hence I have to label my polymer, nucleus and DNA. However, since my DNA labeling kit has not arrived, and I want to train myself using the confocal microscope, I started my experiment first using my labeled polymer and nuclear labeling.

The cell line I chose is HepG2, it is a human hepatocellular carcinoma cells, mainly because it does not detached easily from the tissue culture plate. I label my polymer with FITC, and to stain the nuclei, I used Hoechst dye that is specially used to stain ‘live’ cells.

Why use CLSM?

In order to view my cells, Confocal laser scanning microscopy(CLSM) is used. The CLSM used is LSM 5 LIVE, the latest edition for imaging of living cells from ZEISS technology. CLSM uses laser to excite the fluorochromes, unlike fluorescence microscope(FM) which uses fluorescence light.

1)CLSM allows 3D view of the sample, hence if there is an overlapping of different fluorescent signals, only CLSM can help differenciate them.

2) CLSM allows viewing of distinct and sharp images by removing out-of-focus light, unlike FM that usually gives blurry images and some signals cannot be viewed distinctly.

If you wanna know the principle of CLSM and FM, just give me a comment, as it is more like ‘physics’, afraid some of you might not understand.

Ok back to my experiment, I prepared 4 different samples at different time interval: 1, 2, 3 and 4.5 hr, these time intervals are chosen after reading some science papers and also FITC ‘photobleaches’ easily, hence I have to prepare samples at different time intervals. The polymer-DNA complexes are given(transfected) to the cells at 0 hr, while the nuclei are stained with Hoechst 40 mins before viewing under CLSM, and the cells are washed 5 times with colourless DMEM medium before viewing to prevent auto-fluorescence. Hence, that is why i used HepG2, because of the washing steps.:)

One important precaution: sample preparations must be done in the dark, once exposed to light, the fluoresce intensity can greatly decrease, especially for FITC.

GREEN(FITC): polymer ( note: DNA is still bound to the polymer, but cannot be seen as it is not labeled)

BLUE(HOECHST): nucleus

The following pictures are a bit blurry because I magnify them 8-12 times from the original one.

1 hr after Transfection: on the cell membrane

Q: Why do the polymers 'stick' onto the membrane?

A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!

2.5 hr after Transfection: in the cytoplasm



Q: How does my polymer enters the cytoplasm?

A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.

4.5 hr after transfection: in the nucleus!!



Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?

A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.

Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :(
But still, research is FUN! :))))

Nisha
TGo2
0503254E

Week 13 Sharing of experiences in histopathological laboratory

In tissue processing room

• Whole mount preparation
  -Completely cross-sections of a pathological specimen
  -Usually too large for the ordinary cassette and require individual handling prior to machine processing
  -Example: whole mount sections of prostate

Observations of prostate trimming by pathologists
-Material: Prostate cutting tool
-Whole mount is necessary as tumour is located in prostate and can only be seen under microscope.
-For whole mount processing, the timing of fixation will be extended for 41/2
hours (total 211/2 hours) to ensure tissue is thoroughly fixed.
-The dehydration steps will also be extended to ensure proper dehydration due to the size of the tissue.
-Other tissues will not be processed together with whole mount prostate as over fixation and dehydration of smaller pieces will cause the tissues to be harder.


Microtomy

1. Microtome safety (Safe Operating Procedure)
2. Handle blades very carefully when installing or removing. Follow the manufacturer’s guidelines explicitly.
3. Tungsten-Carbide knives can put through your shoes, if dropped. Be careful where your feet are positioned when installing or removing blades.
4. Store blades in a covered container that has guides to hold the blades rigid.
5. Never leave blades on countertops. Lacerations can occur when reaching across the countertop and inadvertently contacting on unprotected table.
6. When setting up the microtome, position the sample first, and then put in the blade. Never the other way around.
7. When applying the brake, ensure that it is tight. Most accidents occur when the brake slips and the operator’s hand is drawn into the blade.
8. When leaving the microtome, even for a short time, ensure that the blade guard is in place.
9. When preparing a paraffin sample for the microtome, remember to clamp the sample down tight. The movement is allowed by a loose clamp increase your risks of cut.
10. Use forceps or brushes during retrieving and transferring slides and ribbons from the blade, thereby keeping your hands free from the moving parts of the microtome.

Common faults encountered in microtomy and remedies

Causes / Remedies

Ribbon fails to form
1. Paraffin too hard / 1. Use softer wax i.e. lower melting
2. Blade too blunt point / 2. Change to a new blade edge
3. The tilt is too great / 3. Tilt the blade less

Crooked ribbons
1. Edge of block is trimmed not parallel to blade. / 1. Re-trim the block.
2. Irregularities in blade edge / 2. Try another part of the blade

Sections compressed, wrinkled and jammed together
1. Blade too blunt / 1. Change to a new blade edge.
2. Paraffin block is warm / 2. Cool the block on cryoplate
3. Blade tilt is too slight, therefore / 3. Increase the tilt
the cutting facet rubs over the block
4. Blade edge gummed with wax / 4. Wipe blade with xylene

Sections crumble and specimens fall out, mushy and soft
1. Incomplete dehydration / 1. Re-hydrate
2. Improper embedding / 2. Re-embed

Tissues turn hard and cut like stone
1. After clearing, the specimen was / 1. Nothing can be done except repeat
accidentally left to dry in air with fresh specimen
2. Paraffin bath is too hot and burns the tissue / 2. Decrease the temperature of parrafin
bath

Split ribbon or lengthwise scratches in ribbon
1. Nicks in the blade / 1. Use a different part of the blade.
2. The tilt of the knife is too great / 2. Decrease the tilt
3. Blade edge dirty / 3. Wipe blade edge with xylene
4. Hard particles (calcified materials) in the block / 4. Do surface decalcification
5. Crystal from fixative e.g. Mercuric chloride / 5. Treat tissues to remove crystals

Undulations in the surface of the section
1. The blade is not tightened / 1. Tighten all screws
2. The title of the blade is too great / 2. Decrease tilt to prevent vibration
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4. Blade edge is dull or rounded / 4. Change to new blade edge

Sections of varying thickness and skipping of sections
1. Blade not tilted enough to clear facet, / 1. Adjust the tilt
or too much and tissue is compressed until
the inevitable expansion gives a thick section
2. The blade and specimen holder not tightened / 2. Tighten all screws
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4.Cutting stroke is not regular or interrupted / 4. Maintain regular cutting stroke

Sections full of fine lines running across
1. Blade edge consist of fine serration / 1. Change to a new blade edge
2. Cutting edge is dirty / 2. Clean blade edge

Sections full of folds
1. Water bath is too hot or too cold / 1. Adjust temperature to 50 degree
Celsius
2.Blade tilt is too slight so cutting facet / 2. Increase the hilt
rubs over the block.
3. Paraffin block is warm / 3. Cool block on cryoplate

Alright i shall stop here then. :)

Ang Xiao Si Sharon
TG02
0503219H

Week 12 Attachment Sharing

Hey guys! 12 weeks have passed. I have started with a second project that covers expression proteomics of a clinical isolate of Stenotrophomonas maltophilia, a nosocomial infectious agent. Expression proteomics relates to studying the entire secretory profile of the bacteria. In my case, i will be precipitating the secretory proteins from S. maltophilia grown at both 28˚C and 37˚C.

Methods
1) Isolate is streaked on an LB agar plate and incubated at 37˚C for 24 hours
2) A colony will be inoculated into 20mL of LB broth and incubated at 28˚C for 24 hours
3) 5x10^7 cells will be inoculated into 12 tubes in which 6 will be incubated at 28˚C and the
other 6 will be incubated at 37˚C
4) Secretory proteins in the supernatant after centrifugation will be transfered to teflon tubes
5) Equal volumes of 40% TCA in Acetone as the supernatant will be added to precipitate the
proteins
6) Tubes will be incubated at 4˚C for 1 hour and inverted every 10 mins
7) Tubes are then centrifuged at 16,000xg for 1 hour at 4˚C
8) Keeping an eye on the protein pellet, supernatant is decanted
9)Remaining supernatant is aspirated out and 250uL of pre-chilled Acetone is added
10) Acetone is used to wash the proteins
11) Washed protein is transfered to a fresh Eppendorf tube and centrifuged
12) Supernatant is aspirated and pellet is allowed to air-dry
13) 200uL of Rehydration Buffer is used to rehydrate the protein pellet
14) 2uL of Protease inhibitor was added to prevent denaturation of proteins
15) Bradford Assay is performed to determine the final protein concentration
16) If there is sufficient protein sample, a 2-Dimensional gel can be run
17) Spots containing significant proteins can be identified and excised
18) Protein spots are then analyzed through the MALDI TOF/TOF
19) Peptides can then b identified through the Mascot Search program

Chemicals
That was a brief itinery of what is carried out in my experiments daily. A single experiment can last for up to 2 weeks. Now i'll explain about certain chemicals that are required for protein precipitation.

Acetone is used for the washing of protein pellets after precipitating with 40% TCA in Acetone. Acetone is the simplest and most important of the ketones. It is a polar organic solvent and therefore dissolves a wide variety of substances. It has low chemical reactivity. These traits, and its relatively low cost, make it the solvent of choice for many processes. About 25% of the acetone produced is used directly as a solvent. Acetone is also used as a drying agent, due to the readiness with which it binds to water, and its volatility. Also, by washing with Acetone, any impurities that have accumulated in the process is removed from the proteins.

Since Acetone can act as a drying agent, the protein pellet will be rid of most liquids thus Rehydration Buffer is required to rehydrate the proteins. The Protease Inhibitor is used to prevent any proteases from acting on the precipitated proteins. Proteases may still be present because they ar enzymes which are also proteins.

Aseptic techniques
When working with proteins to perform analysis, it is important to not come into contact with the samples since it can contribute to the quality and quantity of proteins analyzed. For example, if we do not wear gloves and come into contact with the sample, keratin may be found abundantly in our protein sample. If we talk into our tubes during air-drying, enzymes may be added to our samples.

Conclusion
Thus, i conclude by saying that even though research may seem easy to some and boring to others, i have to say that they are very wrong. I have enjoyed my 12 weeks at the research centre so far through all the ups and downs and all the "failed" experiments. It is inevitable not to get good results but they ARE results so we learn from them. This is the end of my second entry after 12 weeks. Hope to blog in 6 weeks time with more interesting facts to share. See you guys at the next Campus Discussion!

Johanna
0503309G
TG02

Week 11 Attachment Sharing

Heya guys....how are all of u doing......???11 weeks have passed.......9 more weeks to go...dunnoe if that's good news or bad news....8-?......ahkahkahk....aniwei this time round....i would be blogging about something all of us are very familiar with.......PCR!!!.......

Title: PCR of subjects' DNA (24 for Chinese, Malays and Indians each)

Purpose: To make a lot of copies of the specific DNA fragment that needs to be analysed.

Materials and Methods:

1. Prepare master mix for 75 samples containing the following components:

2. Pipette 8ul of the master mix into each well of the 96-well PCR plate.

*those boxes coloured are filled with the 8ul master mix.

3. Pipette 2ul of the DNA into the wells in the following order:

4. Cover the plate with silicon mat

5. Spin down the plate at 200G for 20 sec

6. Put the plate in the PCR machines. The standard conditions for PCR are:

7. After PCR has finished, spin down the plates at 200G for 20 sec

That's all for now...hope u guys enjoy the last few weeks of your SIP...all da best guys!!!!:)

Nur Zahirah

0503165C

TG02

Week 10 Attachment Sharing

Hi guys!! It is my turn to blog again! For the past few weeks I have been handling 2 projects on allele discrimination. As mentioned previously in my blog, I used allelic discrimination kit to identify the different alleles present in the samples provided. However, there is another method to differential the allele, namely the Enzyme-based method. This method involved a lot of molecular biology principle and is quite time consuming too.


Despite that the duration for this method is rather long, the results produced is much more easier to analyze and interpret. Nevertheless, this method is not suitable for most of allele discrimination. It can only be used if the polymorphism of the allele can be ‘coincidently’ digested by any specific restriction enzymes. In my case, it happens that the alleles we emphasized are different by a nucleotide and can be digested by a particular enzyme.



Recipe for Enzyme-based methods

1.First and foremost, the DNA samples are amplified by using the PCR thermocycler and specific primers (both forward and reverse) close to the target gene.
2.The DNA samples then undergoes restriction digestion performed by a specific restriction enzyme (it is very important that the restriction enzymes cutting site matches with the polymorphism).
3. Digested samples are run in gel for separation of alleles
4.Stained the gels in EtBr (Recommendation: add EtBr when casting gels, it is a lot better!!)
5. The gels are then detected under UV light.



Interpretation of results:

Since the restriction enzymes is able to use the polymorphism as it cutting site, only DNA samples that comprised of the allele will be digested. For example if allele y contains the restriction enzymes cutting site (contains the target nucleotide), restriction enzymes will be able to cut the allele into two fragments. In contrast, if it is allele Y then there will be no cutting site for the enzymes to act on. As a result, allele y will appear below allele Y after electrophoresis. This is because allele y has been digested into smaller fragments and hence will drift faster than allele Y in the gels. Then again if the DNA samples is a heterozygous, there will be a double bands. Since only one of the chromosomes contains the cutting site, this amount of chromosomes digested will also be half while the rest remain uncut. Hence, a double band will appear as half of the digested fragment (digested one) drift faster than another half (undigested).



Other methods for allele discrimination:

Dynamic allele-specific hybridization (DASH)
Basically this method worked by measuring the difference in melting temperate in the DNA samples. Unlike the other methods, DASH used biotinylated primer with a bead attached to it. The biotinylated primer with the bead is then extend the DNA samples is amplified.
The amplified DNA samples is then incubate in streptavidin column. NaOH is then added to wash away the unbiotinylated DNA. This is because the unbiotinylated DNA does not have the bead joined it and attached to the column. An fluoresces allele specific oligonucleotide is then added to form hybrid with the amplicon. Last but not least, heat is given to determine the Tm of the DNA samples. If there is present of single nucleotide polymorphism the Tm will be lower.


PCR-based methods

In this method, it used two pairs of PCR primers. Each of these primers are specific to their respective alleles.The primer consist of the single nucleotide polymorphorism.Since each pair of primers anneal specifically to their allele. When undergoes PCR process only either one of the primers will bind to the target DNA and amplified.



That all for the week!! Hope your guys have fun and feel free to give comment!!


TG02

Avery, May Lee ( 0503292E)