Greetings to everyone, :)
My previous post was about slide-making, the process of dropping cells onto the slides for observation of chromosomes under phase contrast microscope. I will be explaining the process of slide-staining after dropping slides.
Contents of my post
1) Method for slide-staining
What is slide-staining?
It is the process of staining slides to observe chromosomes under microscope.
Materials for Slide-staining
Reagents
Wright’s stain - 8ml
7.5% sodium bicarbonate solution - 3-8 drops
Hank’s Balanced Salt Solution 1X - 80ml
Giemsa’s azur-eosin-methylene blue solution - 10 drops
0.25% Trypsin Solution - 8ml
Buffer Solution pH 6.8 (1 Merck buffer tablet with 1L of pure water) -40ml
Depex mounting medium - 10ml
Materials
Coplin Jars - 4 jars
Slide - 1
tweezers - 1
Manual air pump - 1
serviette - 10 pieces
cover slips - Dependent on the number of slides
Stop watch - 1
Methods for slide-staining
1) The slides were aged at 90 degree C for 90 minutes. While staining, the reagents were prepared using the 4 coplin jars.
2) The 1st coplin jar contained 40ml of HBSS, 8ml of 0.25% of trypsin and 1-5 drops of Na2CO3.
3) The 2nd jar contained 40ml of HBSS and 1-5 drops of Na2CO3.
4) The 3rd jar contained 40ml of pH 6.8 buffer, 8ml of Wright’s stain and 10 drops of Giemsa stain.
5) The 4th jar contained 40ml of pure water.
6) One slide from every single batch was used as a test slide to test for the quality of staining. If the quality was considered acceptable, the rest of the slides from the same batch could proceed with the staining.
7) The slides were placed in the 1st jar for ~30s -35s followed by a few rinses in the 2nd jar. The slides were placed in the 3rd jar for ~4 min followed by a few rinses in the 4th jar.
8) The slides were wiped, dried using the manual air pump and mounted with Depex with the cover slips.
9) The slides were observed under the microscope. The chromosomes were observed at 100x magnification and evaluated based on its morphology.
The purpose of using trypsin in the 1st jar is to degrade certain proteins found on the chromosome. Though the proteins being degraded are unknown, the purpose has been acknowledged. Without degrading these proteins, chromosomes bands would be indistinctive and chromosome would appear dark or pale.
Take note that the trypsin and staining can be adjusted to produce slides which are well and appropriately stained. If bands are under trypsinized, increase the trypsin time and vice versa. If the chromosomes appear pale on the slide, increase the staining time and vice versa.
That would be all, feel free to ask me any questions regarding this topic. I would try to answer them as accurately as possible. :)
Hey Rachel =)
ReplyDeleteCool post =) The trypsin time is quite short, and staining time is quite long. For Pre-natal specimens ?
I'm curious to ask.. How many slides do you all stain in one batch ? If there are subsequent batches, use another trial slide or just increase the timings be a specific time ?
Thanks, Take care rachel =)
Ng Tze Yang Justin
0703747F
hey rachel!!
ReplyDeletei would like to ask if there is any actual amount of times that you rinse your slides? How do you determine if the rinses are enough or not?
I would also like to know if you can reverse a stained slide if it has been overtained?
LOVE YOU RACHEL!
Renee
TG02
0703634F
Sorry for the late replies,
ReplyDeleteTo Justin, :)
I would not say that the difference in the trypsin and staining time is for the prenatal specimens. Such timings are also used for the adult specimens. The timings which I have mentioned are mainly for the blood specimens. I stained approximately 6 - 8 slides per batch. For every batch, i would use a trial slide to determine the trypsin and staining time.
Thanks and take care too !! :)
To Renee,
There isn't an actual amount of time to rinse the slides. I usually would rinse the slide for about 3-4 seconds just to remove the excess trypsin and the stain. I would not usually determine whether the rinsing time is enough because what I am more concern is whether the chromosomes are well stained. If they are, I would not bother about the rinsing time unless the chromosomes are under-stain or over-stained. This is the part where I would adjust my rinsing time. For example, if the chromosomes are over-stained, i would rinse my slide longer ~10-15 seconds. If the chromosomes are under-stained i would just stained them again for maybe about ~1 min. :) Yes, we can reverse the stain by rinsing the slide in water for about 10-15 seconds. :)
Thanks for you LOVE !! :)
Rachel :)