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How to remove coverslips from mounted slides ?
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+ Histological staining
+ Histopathological techniques
+ Histology
+ Immunostaining
+ Histological techniques
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Meseret Simachew Bezabih
How to remove coverslips from mounted slides ?
Hi Samira. Cytoseal is actually toluene based. Xylene should also work, but maybe Toluene would work faster. I find that if they are very old you may need to soak for a week. I hand agitate them back and forth in the solvent each day until the coverslip starts to drop down below the slide, then I wave them back and forth quickly in the liquid until it falls off by itself. If you physically pull them off, the cells or tissue may lift with the coverslip.
Hi Samira. Cytoseal is actually toluene based. Xylene should also work, but maybe Toluene would work faster. I find that if they are very old you may need to soak for a week. I hand agitate them back and forth in the solvent each day until the coverslip starts to drop down below the slide, then I wave them back and forth quickly in the liquid until it falls off by itself. If you physically pull them off, the cells or tissue may lift with the coverslip.
I want to extract DNA from blood smear slides. I am using xylene to dissolve the mounting medium, but the time for the coverslips to come out varies from 3 days to over 20 days. The mounting medium is all the same for the slides-called Cytoseal 60. The slides are over 2 years.
Do you have any ideas about the reasons that cause the difference in the amount of times to dissolve mounting medium and the potential ways to standardize the time, so the DNA extracted from the blood won't be affected too much
I want to extract DNA from blood smear slides. I am using xylene to dissolve the mounting medium, but the time for the coverslips to come out varies from 3 days to over 20 days. The mounting medium is all the same for the slides-called Cytoseal 60. The slides are over 2 years.
Do you have any ideas about the reasons that cause the difference in the amount of times to dissolve mounting medium and the potential ways to standardize the time, so the DNA extracted from the blood won't be affected too much
Guys. I think you all are missing the point. Dako fluorescent mounting medium is aquous polymer based medium. What Emilie Simmonet has done is the right way. She has stained the sections for immunofluorescence and mounted them in an aquous mounting medium for fluorescence microscopy. Soaking the slides in PBS even at RT would dissolve this mountant and relieve the coverslip without damaging the cells/section. Xylol is best used for Organic resin based permanent mountants like DPX, however DPX is not used for fluorescent stained sections.
Guys. I think you all are missing the point. Dako fluorescent mounting medium is aquous polymer based medium. What Emilie Simmonet has done is the right way. She has stained the sections for immunofluorescence and mounted them in an aquous mounting medium for fluorescence microscopy. Soaking the slides in PBS even at RT would dissolve this mountant and relieve the coverslip without damaging the cells/section. Xylol is best used for Organic resin based permanent mountants like DPX, however DPX is not used for fluorescent stained sections.
Mr. Tartor, If you stained your IHC with DAB as the chromogen, don't worry about leaving the slides in xylene for 3 days, DAB will not be affected; hence the stained end product (brown color) will remain the same in intensity. Secondly, no need to re-hydrate. Right after the coverslips are removed, leave for 1-5 minutes in xylene to dissolve hardened mounting medium, then re-coverslip.
Mr. Tartor, If you stained your IHC with DAB as the chromogen, don't worry about leaving the slides in xylene for 3 days, DAB will not be affected; hence the stained end product (brown color) will remain the same in intensity. Secondly, no need to re-hydrate. Right after the coverslips are removed, leave for 1-5 minutes in xylene to dissolve hardened mounting medium, then re-coverslip.
I put old slides (from more than two years ago into) into fresh xylene under gentle agitation, and after 5 days they eventually separated. I replaced xylene with fresh xylene one time, and I also kept adding xylene in case of evaporation. For new slides (two weeks before) it only takes 3.5 hours. After separating coverslip, I kept samples in fresh xylene for few minutes to ensure dissolving precipitate of old cytoseal xyl.
I put old slides (from more than two years ago into) into fresh xylene under gentle agitation, and after 5 days they eventually separated. I replaced xylene with fresh xylene one time, and I also kept adding xylene in case of evaporation. For new slides (two weeks before) it only takes 3.5 hours. After separating coverslip, I kept samples in fresh xylene for few minutes to ensure dissolving precipitate of old cytoseal xyl.
Hi everyone, thanks for your answers ! I tried to incubate the slides in PBS à 37°C overnight, and the day after, the coverslips came off by themselves. I did a counterstaining, which worked perfectly, and the primary staining was still there. So, if the same pb happens to you, you can try it !
Hi everyone, thanks for your answers ! I tried to incubate the slides in PBS à 37°C overnight, and the day after, the coverslips came off by themselves. I did a counterstaining, which worked perfectly, and the primary staining was still there. So, if the same pb happens to you, you can try it !
Soak the slides in xylene until the coverslips are removed. Rinse off residual medium from the tissue sections in 2 changes of fresh xylene; followed by descending grades of alcohol starting from an absolute alcohol (2 changes) ending in 95% (1 change), followed by DI water. Proceed to counterstaining.
Soak the slides in xylene until the coverslips are removed. Rinse off residual medium from the tissue sections in 2 changes of fresh xylene; followed by descending grades of alcohol starting from an absolute alcohol (2 changes) ending in 95% (1 change), followed by DI water. Proceed to counterstaining.
I agree with Arumugam (that Emile is doing the correct procedure for fluorescence aqueous mounting), except in the statement about DPX. Many people do use dehydration followed by clearing in Xylene or Histoclear followed by DPX for fluorescence work. It does work as a substitute for aqueous mounting...with the added bonus of clearing the tissue better and being more of a more compatible refractive index with oil immersion lenses for high resolution imaging.
I agree with Arumugam (that Emile is doing the correct procedure for fluorescence aqueous mounting), except in the statement about DPX. Many people do use dehydration followed by clearing in Xylene or Histoclear followed by DPX for fluorescence work. It does work as a substitute for aqueous mounting...with the added bonus of clearing the tissue better and being more of a more compatible refractive index with oil immersion lenses for high resolution imaging.
Hi Samira. Cytoseal is actually toluene based. Xylene should also work, but maybe Toluene would work faster. I find that if they are very old you may need to soak for a week. I hand agitate them back and forth in the solvent each day until the coverslip starts to drop down below the slide, then I wave them back and forth quickly in the liquid until it falls off by itself. If you physically pull them off, the cells or tissue may lift with the coverslip.
Hi Samira. Cytoseal is actually toluene based. Xylene should also work, but maybe Toluene would work faster. I find that if they are very old you may need to soak for a week. I hand agitate them back and forth in the solvent each day until the coverslip starts to drop down below the slide, then I wave them back and forth quickly in the liquid until it falls off by itself. If you physically pull them off, the cells or tissue may lift with the coverslip.
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keep them in xylene till the DPX dissolved then gently remove the coverslip to avoid underling tissue damage
keep them in xylene till the DPX dissolved then gently remove the coverslip to avoid underling tissue damage
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Lilian Antonio
Hi, Dr. Lilian
I want to extract DNA from blood smear slides. I am using xylene to dissolve the mounting medium, but the time for the coverslips to come out varies from 3 days to over 20 days. The mounting medium is all the same for the slides-called Cytoseal 60. The slides are over 2 years.
Do you have any ideas about the reasons that cause the difference in the amount of times to dissolve mounting medium and the potential ways to standardize the time, so the DNA extracted from the blood won't be affected too much
Thanks
Jia
Lilian Antonio
Hi, Dr. Lilian
I want to extract DNA from blood smear slides. I am using xylene to dissolve the mounting medium, but the time for the coverslips to come out varies from 3 days to over 20 days. The mounting medium is all the same for the slides-called Cytoseal 60. The slides are over 2 years.
Do you have any ideas about the reasons that cause the difference in the amount of times to dissolve mounting medium and the potential ways to standardize the time, so the DNA extracted from the blood won't be affected too much
Thanks
Jia
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VOTE
Guys. I think you all are missing the point. Dako fluorescent mounting medium is aquous polymer based medium. What Emilie Simmonet has done is the right way. She has stained the sections for immunofluorescence and mounted them in an aquous mounting medium for fluorescence microscopy. Soaking the slides in PBS even at RT would dissolve this mountant and relieve the coverslip without damaging the cells/section. Xylol is best used for Organic resin based permanent mountants like DPX, however DPX is not used for fluorescent stained sections.
Guys. I think you all are missing the point. Dako fluorescent mounting medium is aquous polymer based medium. What Emilie Simmonet has done is the right way. She has stained the sections for immunofluorescence and mounted them in an aquous mounting medium for fluorescence microscopy. Soaking the slides in PBS even at RT would dissolve this mountant and relieve the coverslip without damaging the cells/section. Xylol is best used for Organic resin based permanent mountants like DPX, however DPX is not used for fluorescent stained sections.
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Put slide into the cuvette with xylol for 60 minutes.
Put slide into the cuvette with xylol for 60 minutes.
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Mr. Tartor,
If you stained your IHC with DAB as the chromogen, don't worry about leaving the slides in xylene for 3 days, DAB will not be affected; hence the stained end product (brown color) will remain the same in intensity. Secondly, no need to re-hydrate. Right after the coverslips are removed, leave for 1-5 minutes in xylene to dissolve hardened mounting medium, then re-coverslip.
Mr. Tartor,
If you stained your IHC with DAB as the chromogen, don't worry about leaving the slides in xylene for 3 days, DAB will not be affected; hence the stained end product (brown color) will remain the same in intensity. Secondly, no need to re-hydrate. Right after the coverslips are removed, leave for 1-5 minutes in xylene to dissolve hardened mounting medium, then re-coverslip.
More
VOTE
I put old slides (from more than two years ago into) into fresh xylene under gentle agitation, and after 5 days they eventually separated. I replaced xylene with fresh xylene one time, and I also kept adding xylene in case of evaporation. For new slides (two weeks before) it only takes 3.5 hours. After separating coverslip, I kept samples in fresh xylene for few minutes to ensure dissolving precipitate of old cytoseal xyl.
I put old slides (from more than two years ago into) into fresh xylene under gentle agitation, and after 5 days they eventually separated. I replaced xylene with fresh xylene one time, and I also kept adding xylene in case of evaporation. For new slides (two weeks before) it only takes 3.5 hours. After separating coverslip, I kept samples in fresh xylene for few minutes to ensure dissolving precipitate of old cytoseal xyl.
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By embbeding the slide in xylole the canada palsam or dpx will dessolved and can remove the cover slip easly
By embbeding the slide in xylole the canada palsam or dpx will dessolved and can remove the cover slip easly
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immersed the whole slide in a container with fresh zylene until the coverslip remove without damage to the sample.
immersed the whole slide in a container with fresh zylene until the coverslip remove without damage to the sample.
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Hi everyone, thanks for your answers !
I tried to incubate the slides in PBS à 37°C overnight, and the day after, the coverslips came off by themselves.
I did a counterstaining, which worked perfectly, and the primary staining was still there.
So, if the same pb happens to you, you can try it !
Hi everyone, thanks for your answers !
I tried to incubate the slides in PBS à 37°C overnight, and the day after, the coverslips came off by themselves.
I did a counterstaining, which worked perfectly, and the primary staining was still there.
So, if the same pb happens to you, you can try it !
More
VOTE
Soak the slides in xylene until the coverslips are removed. Rinse off residual medium from the tissue sections in 2 changes of fresh xylene; followed by descending grades of alcohol starting from an absolute alcohol (2 changes) ending in 95% (1 change), followed by DI water. Proceed to counterstaining.
Soak the slides in xylene until the coverslips are removed. Rinse off residual medium from the tissue sections in 2 changes of fresh xylene; followed by descending grades of alcohol starting from an absolute alcohol (2 changes) ending in 95% (1 change), followed by DI water. Proceed to counterstaining.
More
VOTE
I agree with Arumugam (that Emile is doing the correct procedure for fluorescence aqueous mounting), except in the statement about DPX. Many people do use dehydration followed by clearing in Xylene or Histoclear followed by DPX for fluorescence work. It does work as a substitute for aqueous mounting...with the added bonus of clearing the tissue better and being more of a more compatible refractive index with oil immersion lenses for high resolution imaging.
I agree with Arumugam (that Emile is doing the correct procedure for fluorescence aqueous mounting), except in the statement about DPX. Many people do use dehydration followed by clearing in Xylene or Histoclear followed by DPX for fluorescence work. It does work as a substitute for aqueous mounting...with the added bonus of clearing the tissue better and being more of a more compatible refractive index with oil immersion lenses for high resolution imaging.
More
VOTE