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OfflineCyber
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Paraffin embedded samples for sectioning.
    #18466022 - 06/24/13 04:23 PM (10 years, 10 months ago)

I have fixated samples, dehydrated with 30%, 50%, 70%, and 95% ETOH then to xylene for the final step of dehydration before embedding in paraffin and arranging into blocks for sectioning.

The blocks were cooled and trimmed then sectioned into slices.

Now I am having trouble finding the right protocol to remove the paraffin from the slide mounted section and prepare it for staining.

I guess my question is should I just use xylene to remove the paraffin and try to stain the section then mount or should I reverse the ETOH process to dehydrate after removing the paraffin then stain and mount?

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OfflineCyber
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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18484523 - 06/28/13 04:29 PM (10 years, 10 months ago)

I am kind of surprised that no one answered this yet.

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InvisibleinskiM
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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18485839 - 06/28/13 09:48 PM (10 years, 10 months ago)

This is not really the methods used by most mycologists, usually if possible fresh samples are sectioned directly, the sections are made on a few different tangents in order to best view the various different cells, usually the mounting reagent is 3-5%K0H or a similar alkaline reagent, the section is placed on the slide in a drop of the reagent and the cover slip is applied, if a stain is to be used it can be placed on the slide so that it comes into contact with the edge of the cover slip then the corner of a paper towel is placed at the opposite edge of the cover slip, this draws the stain under the cover slip and through the sample, if the stain is too heavy another drop of the original reagent can be applied in the same fashion and drawn through with the paper towel, this will dilute the stain reducing the contrast when viewed under the scope and usually this makes it a bit easier to observe certain cells.

Getting good sections by hand takes practice and using dried samples is much harder, the dried material needs to be rehydrated in K0H or similar alkaline reagent before section can be done.

Sorry I can't answer your question as I have never seen any mycologists use those methods of preservation:shrug:


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OfflineCyber
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Re: Paraffin embedded samples for sectioning. [Re: inski]
    #18486983 - 06/29/13 06:12 AM (10 years, 10 months ago)

I find your statement odd that it is not used by most mycologists. Every peer reviewed article I read starts with a statement that the specimen was fixated in xxx then embedded into paraffin in the usual manner. xxx can be 10%NBF, formalin, and occasionally Flemming's weaker solution. I would suspect that most amateurs do not paraffin embed samples but professional mycologists should be familiar with the process as should most students taking histology.

My only issue is finding documentation as to what "the usual manner" is. At this point I have been testing fixating and mounting processes from both medical and botany on wild samples of Agaricus. I have had some successes and some failures.

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InvisibleinskiM
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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18488791 - 06/29/13 04:05 PM (10 years, 10 months ago)

I'm sorry but I have read many peer reviewed papers and have just looked through all of the ones I have with me and none of them use the methods you describe, all of them use fresh or dried samples that have been hand sectioned and mounted in an alkaline reagent, it is the favoured method of amateur and professional mycologists alike.

Your method may be used for some specific genera but it is not common:shrug:

I've found that the best methods to successfully view all of the taxonomically significant microscopic structures of macrofungi is to use fresh samples, it takes practice to perfect all of the different sections but it is fun, I recommend this book, from this you can learn everything you need to know about examining and describing microscopic characteristics.
http://www.amazon.com/How-Identify-Mushrooms-Genus-III/dp/0916422097


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Re: Paraffin embedded samples for sectioning. [Re: inski]
    #18489037 - 06/29/13 05:03 PM (10 years, 10 months ago)

I have that whole set of books as well as many others.

However, you do not get results like this from those instructions





Those are gill cross sections showing the Hygrophorus praetensis below the Hymenium surface.

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InvisibleinskiM
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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18489568 - 06/29/13 07:12 PM (10 years, 10 months ago)

I get very good results using methods directly from that book.
I see your images show the hymenophoral trama but at that resolution it is hard to make out what type of hymenophoral trama that is, they are nicely set out with good documentation:cool:
Some examples linked below, all methods from the mentioned book, sometimes modified by myself.
http://mushroomobserver.org/55302?q=1KyHG
http://mushroomobserver.org/49806?q=1KyHG
http://mushroomobserver.org/45052?q=1KyHG
http://mushroomobserver.org/44888?q=1KyLb


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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18491728 - 06/30/13 10:15 AM (10 years, 10 months ago)

I don't think you need to go quite this far, but this regimen should do the job [from Millipore's web site, user guides]:

Suggested Reagents and Times for Deparaffinization

Xylene                    2 to 3 changes, 5 minutes each
100% alcohol            2 changes, 3 minutes each
95% alcohol            2 changes, 3 minutes each
80% alcohol             3 minutes
70% alcohol             3 minutes
PBS/Tris buffer         2 changes, 3 minutes each

I played around making blocks years ago.  I can't remember much about how this block was processed, other than it was formalin-fixed. 

cyanescens:






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OfflineCyber
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Re: Paraffin embedded samples for sectioning. [Re: Suntzu]
    #18520942 - 07/06/13 04:14 PM (10 years, 10 months ago)

Suntzu,

I have given that a try and it is working much better. I did come across this one that I am trying now for staining


Staining of Paraffin Sections (Fixative: Any general fixative is adequate.)

    Xylene (labeled Xylene I), 5 minutes.
    Xylene (labeled Xylene II), 2 minutes.
    100% ethanol, 2 minutes.
    95% ethanol, 2 minutes.
    70% ethanol, 2 minutes.
    50 % ethanol, 2 minutes.
    30 %ethanol, 2 minutes.
    Water, 2 minutes.
    Stain Sample, approximately 5 minutes.
    Rinse off the stain in running tap water
    35% ethanol, 2 minutes.
    50 % ethanol, 2 minutes.
    70% ethanol, 5 minutes
    95% ethanol 2 minutes.
    100% ethanol, 2 minutes.
    Xylene I, 5 minutes.
    Xylene II, 5 minutes.

Drain off the xylene. Place a small drop of resin on the sections. Then cover the sections carefully with a coverslip.

Edited by Cyber (07/06/13 04:15 PM)

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Re: Paraffin embedded samples for sectioning. [Re: Cyber]
    #18526490 - 07/07/13 06:28 PM (10 years, 10 months ago)

I am going to add a note here. The above process only works if the permanent mounting medium you are using is Xylene or Toluene based. In my case I am using a water based Apathy's Gum and that did not work.

I am going to need to get some thick balsam and diluted it with xylene. Canadian Balm and Xylene is a permanent mount resin with good optical qualities.

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