2 replies to this topic

[bullfrog]

These days I’m keeping in trying cryosection on Arabidopsis tissue and now it drives me crazy. The idea is like this: we have developed several GFP markers expressed in Arabidopsis inflorescence and we would like to get a clear view on it in terms of cellular distribution pattern. So only sectioning can do that.

I read a few papers where similar experiments were described and I followed the procedures.
The tissue was harvested and fixed with PFA, followed by a series PFA/sucrose solution change until sucrose concentration reached 30%.
Then the tissues were embedded into 4% low melting agarose (around 35C), and the tissue block was trimmed and fixed on cryosection tissue holder with OCT.
The tissue block was then flash frozen with liquid N2, and loaded onto cryomicrotome (temp setting as -20C), slice thickness was 50 micro-meter.
After cutting, the slices were placed on microscope slides and viewed under confocal microscope.

I have two prblems:
1. The tissue usually cracked when the blade was cutting through during sectioning. But anyway, I could still get a good slice from an average of five tissue blocks
2. [major one] NO GFP signal..I dissected some tissues prior to agarose embedding and check, the signal was still there. Therefore I’m very sure that something went wrong during cryosection…

I desperately welcome any suggestions. Thank you very much

[MacJoseph]

bullfrog, on Sep 17 2009, 09:20 AM, said:

These days I’m keeping in trying cryosection on Arabidopsis tissue and now it drives me crazy. The idea is like this: we have developed several GFP markers expressed in Arabidopsis inflorescence and we would like to get a clear view on it in terms of cellular distribution pattern. So only sectioning can do that.

I read a few papers where similar experiments were described and I followed the procedures.
The tissue was harvested and fixed with PFA, followed by a series PFA/sucrose solution change until sucrose concentration reached 30%.
Then the tissues were embedded into 4% low melting agarose (around 35C), and the tissue block was trimmed and fixed on cryosection tissue holder with OCT.
The tissue block was then flash frozen with liquid N2, and loaded onto cryomicrotome (temp setting as -20C), slice thickness was 50 micro-meter.
After cutting, the slices were placed on microscope slides and viewed under confocal microscope.

I have two prblems:
1. The tissue usually cracked when the blade was cutting through during sectioning. But anyway, I could still get a good slice from an average of five tissue blocks
2. [major one] NO GFP signal..I dissected some tissues prior to agarose embedding and check, the signal was still there. Therefore I’m very sure that something went wrong during cryosection…

I desperately welcome any suggestions. Thank you very much

I don't know about the GFP, maybe it is deactivated with the PFA or something. You can try to use anti GFP antibody in addition.
Now, for the cracking of the tissue, 50um is way, way to thick! That is used for trimming. Best results, try 10-20microns thickness. These are better for antibody penetration, also. Your knife probably is not sharp enough, try to sharpen it and don't cut so thick sections.

Hope it helps.

Best,

[krish123]

Sucrose is made by diluting a 50% stock (made in water) with 1XPBS to control the pH.



4% paraformaldehyde is made just like in the in situ protocol. However I save 45 mL aliquots in 50mL conical tubes frozen at ­20šC. Then I microwave for about 30sec to thaw, ice, cool to near 4š, then use.



None of the reagents I use have been treated in any special way to remove RNAse.  I think the fixed cells are enough of a barrier to RNAse getting in and degrading RNA. If you leave out the sucrose steps, nothing will work well - In situs are almost blank and lacZ activity is very low.  This will also happen if you don't do the sucrose for long enough.



I've used this same protocol with success all the way up to 9 month old animals.  The longest I ever fix is 4 hours since overnight fixation tends to kill the lacZ activity in the most exposed regions.



This protocol is based on that in the Z/AP Cre reporter paper from Nagy lab (Lobe, et al., Dev Biol. 1999 Apr 15;208(2):281-92)



Lacz activity in unfixed/unsucrosed tissue frozen in OCT gives extremely faint staining that bleeds out of cells expressing it. The best way I've found to solve this problem is to lightly fix the tissue (too long kills LACZ) then equilibrate it into sucrose before freezing in OCT. This

seems to give excellent lacZ activity, retains it in the cells, and gives good antibody staining and in situs all at once.  The signal for in situs has been about 5X stronger than just frozen tissue.



Below is a reccomendation for a LACZ antibody: We ordered it but I haven¹t tried it. Using a different antibody of Seung Kim¹s lab, I found that X-Gal staining was a much stronger assay.
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