Hi Kishlay:

The first recomendation is, if you can, change the microscope slides by genlbond and forget to prepare slides for ever.

If you can not do it, I recomend melt NMPA in a staining jar (avoid boil) and dip slides in a vertical position, drain them off excess agarose, wipe the back clean and dry by leaving on a horizontal clean bench overnight, remember to hold the agarose hot. After that you can store all together in the original slides box. Remember to keep previously the slides in ethanol in order to degrease them, it may be causing the detachment of the agarose during the experiment. With this way is easy that the agarose become dirty with lint or paper's fibers with which the slides were cleaned.

Another alternative is to deposit 150uL of NMPA over the slides and spread it with the tip of the thumb.

Sometimes the agarose layer does not stick well or retract, you must to degrease the slides better or just throw them away this slides.

Good luck!!

I am having the same problem. Do you use a special brand of slides? Or does any brand work for you?  Thanks!

Hi Karen

It do happens most of the time. I am now answering my own question based on my experiances. I am using slides frm Leica (i can provide you more details if u need). It is best to use Gel bond film as I have experianced. To my experiances I have  never have problem with Gel bond film. And once it is fixed with Ethanol you can preserve  the film. 

Due to some limitation I have to work on with slides. As per my expeiance, every time you use (boil) NMPA and LMPA, there is increase in the concentarion. I would advise not stick with same time you use for solidyfying the agarose. Remove the coverslip from NMPA layer before it is completely solidified (give 3-4 mins, you can feel the diffrenece while removing the coveslip for solidyfying time of 3-4 min and 15 mins). Same applies for LMPA, howver, because it takes more time for LMPA (8-9 mins). Try this and let me know. I hope this work. Also, do not work in high humid environment.

Let me know. Good Luck.

Thanks 

ummmm

Use silanated glass slide! Dip the silanated slide into agarose solution and keep the solution at 70C

Dear Kishlay Kumar ,

Preparation of base slides

1. Prepare 1% (500 mg per 50ml PBS) LMPA and 1 % NMA (500 mg per 50 ml in Milli Q water). Microwave or heat until near boiling and the agarose dissolves. For LMPA, aliquot 5 mL samples into scintillation vials (or other suitable containers) and refrigerate until needed. When needed, briefly melt agarose in microwave or by another appropriate method. Place LMPA vial in a 37ºC dry/water bath to cool and stabilize the temperature.

2. Dip the slides in methanol and burn them over a blue flame to remove the machine oil and dust.

3. While NMA agarose is hot, dip conventional slides up to one-third the frosted area and gently remove . Wipe underside of slide to remove agarose and lay the slide in a tray on a flat surface to dry. The slides may be air dried or warmed at 500C for quicker drying. Store the slides at room temperature until needed; avoid high humidity conditions. We generally prepare slides the day before use.

NOTE: Slides should be labeled before storage.

To the coated slide, add 75 µL of melted LMPA mixed with ~5-10 µL blood sample . Place coverslip and put the slide on a slide tray resting on ice packs until the agarose layer hardens (~5 to 10 minutes). Gently slide off coverslip and add a third agarose layer (80 µL NMPA) to the slide. Replace coverslip and return to the slide tray until the agarose layer hardens (~5 to 10 minutes). Treat slides in Lysing Solution for at least 2hour at ~4ºC and continue the procedure.

Best regard

Assis. Prof. Dr.Wiaam A. Al-AMILI /University of Baghdad- Iraq