Preparing Frozen Tissue Sections

taotan

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Nov 16, 2015
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#1
I have some questions about 'Preparing Frozen Tissue Sections'. First, If I should immerse the sample into the O.C.T. and freeze the sample in to liquid nitrogen ? second, I want to detect a trans-membrane protein , which I can used to fix the sections, 4%PFA or Acetone? Finally, Is there anybody could give me a protocol about the frozen section.Thanks!
 

pundir

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Nov 16, 2015
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Hi Taotan,



here i would like to share a web resource which will really be helpfull for any of your querry related to frozen tissue sections pathologyinnovations.com/frozen_section_technique.htm



regarding you present querry i would add that you can also first fix your tissue in 4%PFA and then section it, for that you may follow the procedure as: overnight fixation in 4%PFA (also depends on tissue thickness) then in sucrose gradients(cryoprotection) 20% then 30 % till tissue sunks to bottom (saturated) then put the tissue in chuck (platform) and add OCT ,freeze quickly in dry ice or liquid Nitrogen and start sectioning.................hope it helps you, for any other querry do post again

 
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#3
Hi Taotan, to answer Your question accurately I would like some more information , what is the tissue , what stain do you want to use.

Yours Oct
 

taotan

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Nov 16, 2015
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#4
Hi pundir, Thank you very much! But I still have a question, need I rehydrate the sections when I detect the my protein just like that in paraffin sections? And How long should I leave my sample in sucrose gradients? Yours Tao
 

taotan

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Nov 16, 2015
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#5
Hi OCT,

Thanks a lot !We want to detect a trans-membrane protein in ovary with immunofluorescence staining.Yours Tao.
 

pundir

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Nov 16, 2015
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#6

Hi Taotan,

as i had mentioned earlier the tissue had to be kept in 30% sucrose till it sunks to bottom, regarding rehydration there is no need, as these are frozen sections you just proceed with your protocol, if your sections are free floating (taken in well plate with PBS/TBS) you can proceed after sectioning but if sections are taken on slide you have to wait for some time so that sections get adhered to the charged or subbed slides and if you are storing sections in -80 C after sectioning then before proceeding for IHC you must bring the sections to room temperature to avoid detaching from the slides.............
 

Biju

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Nov 16, 2015
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#7

Hi

I have seen people using OCT for frozen sections after placing the tissue in chucks and then spray with OCT . This was for small ocular specimens.

Biju
 

taotan

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Nov 16, 2015
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#8
Hi pundir,

As you mentioned , I should treat the sample with 20% and then 30% sucrose solution. I want to know how long should I leave my sample in 20% sucrose solution and then transfer samples into 30% solution. And some guy told I need to do the antigen retrieval, do you think is it necessary? Yours Tao
 

pundir

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Nov 16, 2015
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#10
Hi pundir,

As you mentioned , I should treat the sample with 20% and then 30% sucrose solution. I want to know how long should I leave my sample in 20% sucrose solution and then transfer samples into 30% solution. And some guy told I need to do the antigen retrieval, do you think is it necessary? Yours Tao



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Hi Tao,

you have to leave tissue in sucrose till it sunks to bottom, and antigen retrieval(AR) depends on your antigens epitope free or blocked because of fixation , its not necessary that you always need to go for antigen retrieval, so you first try without AR if it doesnt work then go for AR.............
 

taotan

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Nov 16, 2015
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#11
Hi pundir,
Thank you very much! Because our sample is too difficult to get, I have so many questions. I will try the method you told me as soon as possible. Yours Tao
 

Biju

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Nov 16, 2015
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#12
Hi Taotan

This is how I have seen using OCT in a ocular pathology lab( The specimens are very small to start with.) So spraying might be sufficient to allow frozen sections. I have seen them keeping the tissue at at -80[sup]0 [/sup]freezer for some time and then directly placing on the chucks and immediately spraying with OCT and then sectioning.I want to remind you also that I do not have any personal experience in using OCT for frozen sections.

I feel that you should try different combinations in your lab and see what works best for you.

Hope this helps.

Biju
 

taotan

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Nov 16, 2015
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#13
Dear pundir,
I made a big mistake! I used ddWater to prepare the 20% and 30% sucrose solutions. Will this have severe effects on my sample.Yours Tao