Which glass slides are you using?  Charged slides usually work best.  Also, if you are using a water bath to smooth out wrinkles, it helps to let the slides dry overnight before use to help the tissue adhere to the glass.

I am agree with Eric Sc, please, check the glass slides which you use. It should be adhesive enough (special treatment of glass, a coating or a charge). And it is a good idea to let slides dry overnight before following steps.

Hi...

       I have tried with both gelatin coated slides as well as egg albumin coated slides still the problem is persisting and i am using water bath with temperature 55-60 C. and also i have used over night dried slides only... 

Hey,

If you use regular paraffin you don't need to do it overnight. You just need to melt the paraffin at 60 °C for 1h, then transfer to Xylene and well go away within 5 min.

If the section also goes away use SuperFrost glass slices. That works the best.

also check the adhesive you are using to adhere the section to the glass slide

might be some problem with the dehydration process of the sample. If you are using water bath and proper glass slides and drying them up at 37-42 C...it seems ok, I guess the problem might  be on the sample processing steps

I too suspect it may be your processing.  How do the sections look and cut when you section them?  There may not be enough wax embedded to attach to the slide.  Take one, melt the wax, put back into the clearing agent and look to see what happens.  If it clears, then there just wasn't enough wax (possible some alcohol left over in the specimen to prevent full wax exchange).  The specimen should be totally transparent bey the end of clearing.  If not ( if you see a thin covering layer separating the specimen from the solution) then you still have some water therein.  Just plop back into alcohol (100%) and get rid of that.

another important thing is when you put the section into wax at specific temperature, temperature is very critical factor, because more temperature will damage the tissue

This looks like a processing issue. When you cut does the paraffinstay around the tissue or you get gaps/holes.

What is your processing protocol? How big are the tissue pieces?

Best,

Ivan

Hi Ivan.

       Paraffin around the tissue is there the tissue alone coming out.

       Mid brain of the rat..   I am give five hour hydration and dehydration, 24 hours in chloroform  and each one hour in three wax.. 

Hello Mohamed,

for wax melting 60-65 C and for section ironing 55-60 C.

Seconding Matthew's thoughts.

You should think about (and perhaps tell us) about your dehydration processing too. And brain tissue concerning adherence to slides sometimes isn't quite unproblematic as usually thought.

It might be a bit laborious, but would like to point you to the following threads in RG, if you are interested reading comments on same or similar matter:

https://www.researchgate.net/post/Im_having_difficulties_with_tissue_adherence_to_an_already_treated_slide_Any_suggestion

https://www.researchgate.net/post/Does_sloughing_off_of_cells_in_patches_during_staining_indicate_insufficient_cell_fixing

https://www.researchgate.net/post/What_are_alternatives_to_using_gelatin_as_a_coating_for_adherent_cells_on_glass_slides

https://www.researchgate.net/post/Protocol_for_the_Preparation_and_Chromogenic_IHC_Staining_of_Paraffin-embedded_Tissue_Sections

https://www.researchgate.net/post/How_would_I_prevent_detachment_of_tissue_section_during_the_process_of_immunohistochemical_staining

https://www.researchgate.net/post/What_might_be_the_reason_for_detachment_of_processed_tissue

https://www.researchgate.net/post/How_can_I_avoid_the_loss_of_samples_while_Im_cutting_at_the_cryostat2

https://www.researchgate.net/post/Mouse_brain_slices_30_vs_50_microns_or_higher?

and as an extern link  (general Protocol for embedding into Paraffin) I would suggest:

http://protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/

Good luck too

To add to Matthew's comment.  I also have an intermediate stage of 50:50 xylenes/paraffin mix (in the oven).  Histoclear is a non-toxic version of xylenes. That adds an extra 1/2 hours or so.  I have also learnt that to embed under vacuum (ie:use a vacuum oven), and this really helped the wax move it.  The few times I did not do that, the specimen wasn't as well impregnated.

Try using Superfrost (Fisherbrand) slides.

Brain structures as well as fat tissue need relatively long processing. The chloroform can precipitate your proteins in a much more efficient way compared to alcohols, thus hampering the infiltration. Also limitation can be the fixation. If the samples are not fixed properly, you will have unproper processing. 

What we use as a standard in brains and large fat pieces in hour Leica ASP6025 processor is:

After 48h 4% buffered formaldehyde we store the tissues in 70% ethanol at 4C.

The the program is the following:

Ethanol 70% 1h 37C

Ethanol 85% 1h 37C

Ethanol 100% 1h 37C

Ethanol 100% 1h 37C

Ethanol 100% 1h 37C

Xylene 45min 37C

Xylene 1 h 37C

Xylene 1 h 40C

Paraffin (Paraplast) 1h 60C Vacuum

Paraffin (Paraplast) 1h 60C Vacuum

Paraffin (Paraplast) 1h 60C Vacuum

Works for us.

agree with Susan, it may be your processing. Make sure you have enough wax in the tissue. Adjust wax embedding time according to tissue type and size.

Hello Matthew,

                         Thanks a lot for suggestion really ti could be helpful to me.

                        I used ethanol as dehydration agent. Which dehydration reagent is best among ethanol and iso propanol ?

I think  problem is in  fixation and   tissue processing procedure.

All good answers to this particular issue. But also, yo may wish to consider using an adhesive such as APES or poly-l-lysine on the slides prior to adhering the section from the water bath. Of course, the processing techniques is vital and timings and various optimal solutions are important to consider. Good luck

A lot of good suggestions.

Agree with most, that cause is poor impregnation, which basically could be the result of poor clearing following inadequate dehydration.

I have found a pre-processing overnight stay in 60% alcohol is very helpful and we use isopropyl alcohol without any problems. Clear until the tissue is actually clear. long enough stay in sufficient changes of paraffin. 2 should suffice.

It may be  worthwhile  to begin with a set of fresh solutions.

It might be due to over heating of tissue during wax processing

Have you albuminised the slides while picking up the sections from the water bath ?

Mayer's egg albumin smearing just before mounting the tissue over the glass slide will enhance the adhesiveness of tissue  

Hello!

...if the paraffin processing is good, you can try  the adhesion method of slides with aminoalchilhidrossixylane a chelant solution, that created an  elettrostatic bound between a silical molecular of the glass and ion of the tissue elements.

I'll trying to explain the method:

-The slides in the rack was immerged into the 5% solution water + soap tensioactive for 1 hour

-Rinse under run water to 3 from 4 hours so you'll make a quickly last rince with distillated water.

-Drying the slide on oven at 37°C overnight

-At the day after you may begin the adhesion treatment in this way:

You must  be prepared 5  jars .

The first jar  cover with 2% solution of AES Sigmaldrich+Aceton,  so immerged the slides with the rack for 2 minutes; thereafter in  the second, in the third and in the fourth jar, that were covered up  distillated water,the slides are rinsed in quick succession into each of jar.

Drying the slides into the oven at 37°C.

At the end you must have a good adhesion of the section.

Unfortunately this method is to make up too much time but it's a  good either Histology, IHC, and IF. 

Differently  you can buy elettrostatic  slides, that are better for immunohistochemistry anyway.

I'm advice you another slides adesion method so that you can employ during the cutting of the samples with microtome.

The solution  is be called Biopath (glycerin+cromallumen) that  may be put into the water bath at 37°C  at 1:100, .

That are all my advice, if and only if you carried on a good samples paraffin embedded.

Good work

silvia

hello,

It can be from  parafin bath or uncorrected blocking. Change parafin with new parafin.

Go through every step of processing carefully. Make sure about temperatures. Select the right type of paraffin wax with ceresin

Proper Impregnation after proper (not too much) dehydration is essential. I think even if it is longer/changes impregnation in wax is OK.

Processing is a key step. Depending on the tissue you are handling. I used to have this problem especially handling tissue that has low support architecture, eg. Spleen of fish. The tissue often doesnt stand the same process protocol you use for tougher tissue. Its always better to process like-tissue types together. Another probable reason in wax imregnation. Softer tissue needs less dehydration and low exposure to xylene/clearing agent.