In our lab, we boil pre-cleaned slides in water using microwave (1-2 mins), wipe off the water and then coat with warm normal melting point agarose (NMPA) and air dry for 48 hours and continue with the assay. It has always worked for us. 

Dear Chhaya Godse,

Please brief your problem more.  For what purpose you need clean slide? and what are the problem you are facing?

 As i understand, you need it for comet assay and you might be face gel adherence problem. Therefore, here i am sharing my experience regarding this issue;

We are doing both alkline and neutra comet assay in our lab since 2012.

Earlier we have faced the same problem in slide preparation and agrose gel washout  from slides after electrophoresis.

But after standarisation, now we can confidently do it (40-50 slides in a day).

Few things which we are strictly follow:

1. Use of frosted slides

2.  Wipe with methanol followed by tissue paper. ( make sure slide shouldnot be oily/greasy)

3. Put the slides on hot plate 50-60 C before precoating with NMA and keep at same temperate till completely dry.

4. Store the slides at  dry and moistre free environment till use.

 For more info you can follow the following article 

kumar el al 2015 mutagenesis

link: 

https://mutage.oxfordjournals.org/content/early/2015/04/08/mutage.gev026.full.pdf+html

https://www.researchgate.net/publication/274781214_Sesamol_attenuates_genotoxicity_in_bone_marrow_cells_of_whole-body_-irradiated_mice

Arun Kumar

Article Sesamol attenuates genotoxicity in bone marrow cells of whol...

Hello Chhaya,

The quality of slides is very important for comet assay. To start with, it is advisable to use new 1/4th frosted end slides from a  reputed company for each run. We usually boil them in soap water for 2 minutes in the microwave, wash them gently with soap using a soft brush to make them grease free without any scratches, dry under the fan, dip in methanol, flame and burn off the methanol and then proceed to the first layer step. This usually gives best results with even agarose layering and a clean background during image procurement.

I hope this helps.

Thanks Ilika! I will add the soap cleaning step in our slide preparation. That's good suggestion

Hi Chhaya, 

Did the cleaning protocol work for your assay? I have just started preparing slides for COMET assay and I am finding that after leaving the first 1% NMA layer to set and dry, it starts to pull away from the edges and shrinks, such that the slide looks clear.

I have washed a couple such slides thinking there is no agarose layer left on it but there is still a layer I was surprised to find. Has this happened to you? Could you please post a picture of your 1% NMA coated slide?

Thanks

Prachee

It require attention to distinguish between coated and without coated slides after drying process. 

Close observation of slide will definitely help you to observe the agrose layer on slide.

Here few pics of coated and without coated slides for your reference. 

Hello Arun, many thanks for your slide pictures.

I have attached one of my slide pictures to show you. I have made a mark on the slide upto where the agarose layer was. And as it has dried, you can see the 'shrinking' of the agarose. What is happening here please? Does the layer go transparent as it sets and dries or is the agarose 'evaporating'??!! Or do you reckon the slides need extra cleaning? This is 1% NMA.

Really appreciate all your help. 

Hello Gokhale,

Thanks for sharing picture of slide made by you.

It seems that you have clicked it during process of  drying (half dry). 

During drying process it  looks like that gel is shrinking but its not so. Don't worry, you have to wait till complete dry of slide. 

 After dry, layer go to almost transparent.

After complete dry for conformation, you can screch  both ends of the single slide upto where you coated.

Make sure drying process should be on hot plate  and temp arround 50-60 degree centigrade.

slides should not oily and drity. If it is so you will face difficulty to spread the agrose and your gel will remove further during lysis or eletrophoresis.

hope it will help you . your further query regarding this is most welcome.

Good luck.......

Hello Prachee

We also observe same phenomenon during drying process; but slides are fine after complete drying and do not affect assays... as rightly described by Arun Kumar

Please refer the attached file where you can see the applications of Micronucleus Assay, Chromosomal Aberrations and Comet assay. 

Article Antigenotoxic Potential of Rutin and Quercetin in Swiss Mice...

Hi researchers,

 I have 2 major problems about comet assay.

first problem, after 1 day dry period  I get the slide. is there any suggestion about the problem ? (Figure)

how can I solve the problem ?

second problem

adherence problem about the NMA  after lysis or electrophoresis

I try to Arun Kumar suggestions but  it is not solve the problem.  Is there any  suggestions?

Dear Sedar Yedier,

1. problem:

As i can visualize your slide in image, its shrinking of gel. Make sure that during pre-coating of slide with NMA your slides should be kept on Hot plate ( 50-60 C) till dry. Please put off the Air-conditioner/fan of the room.

2.problem

 Please ensure that your slides should not greasy or oily before pre-coating. wipe the slide with methnol swap followed by dry tissue. You can confirm the oil free surface during precoating with smooth or equal distribution of NMA on slide.

slide should completely dry on hot plate

storage of slides at dry and moisture free box.

Before using just pre-warm the slide on hot plate to evaporate any moisture on it. 

In our's study for lymphocytes 1 hours lysis is sufficient 

handle the slides with most care during lysis -unbinding-electrophoresis- neutralizing-fixing/staining.

for safer side prepare two slides for each sample.

Best Wishes and Good Luck!!!

Regards

Arun

I use NaOH and it works; it cleans and removes the wax layer

Hi everyone! It was actually a manufacturing problem (defect) in those slides we were trying back in the day. Some brands have the same "defect" and are useless for the comet assay. Nowadays I only use Leica slides, and they already come clean, ready to work with. That was our best solution.

Just by using 5M NaOH solution, you can clean your slides perfectly.

Hello everyone,

I agree with Arun's suggestions and thank you for the pictures of the slides. I have also noticed that formation of snowflake-shaped cracks (as shown in the picture shared by Sedar Yedier) on the 1st layer happens when there is any salt contamination in 1% NMA solution. I usually prepare small amounts of 1 % NMA solutions in double distilled water usin clean spatula and glassware, seal with parafilm and store it in the refrigerator till it is exhausted. It is better to prepare it freshly and avoid long term storage. Moreover, slides should be reused as less as possible for proper layering.

Good luck!