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Author Topic: Pollen contaminated frozen sections???  (Read 6494 times)

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Offline Erebus

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Pollen contaminated frozen sections???
« on: August 08, 2006, 07:35:20 AM »
Hi,

Has anyone experienced/heard of frozen sections contaminated with pollen(reproductive cells of plants)?

Pollen contaminated frozen sections???
« on: August 08, 2006, 07:35:20 AM »

Offline excalibur

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Pollen contaminated frozen sections???
« Reply #1 on: August 08, 2006, 04:50:54 PM »
Does your building have any open windows or are the doors near your lab?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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Re: Pollen contaminated frozen sections???
« Reply #2 on: August 09, 2006, 01:58:18 AM »
I haven't heard of it but you have peaked my curiosity ... do tell us more details![/color]

Offline Erebus

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Pollen contaminated frozen sections???
« Reply #3 on: August 09, 2006, 04:23:37 AM »
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Does your building have any open windows or are the doors near your lab?


Answer: Open window in near vicinity of cryostat.  8)  

Quote
I haven't heard of it but you have peaked my curiosity ... do tell us more details!


I am currently scanning sections of rat brain for xenografted stem cells. For this i use various staining procedures, enzymatic/fluorescence. The rats were sacrificed one year post transplant, so i am prepared for all kinds of outcome, despite my open mind this finding really confused me  :shock: .  

I was testing my new primary ab, human nuclei(huN, chemicon), with alexa 488. At first things looked as expected, apart from this "lump".

Description of "lump":
A spherelike collection (15-20) of cell nuclei (10-20 micron diam.) undergoing mitosis.

The thing is:

1. "Lump" emits all wavelengths produced by my fluorescence filter set. Weakest emission in alexa488spectrum...  ...strongest somewhere red (fiercely red).

2. "Lump" not exactly aligned with tissue nearby, not significantly non-aligned though.

3. Almost all "Nuclei" in groups of four. Not a coincidence.

Found a picture of autofluorescent pollen grain. The tetrastructure is similar to the "nuclei" in groups of four.


Question is: Is it even remotely possible that a lump of pollen grain could survive the iterated washing procedure?

My guess is red blood cells/pollen grain. I will do enzymatic staining on consecutive slides just to make sure...

I have pictures of the "lump". Need to work out a way to post them. Maybe i can email them to you? Check out my avatar...

Offline excalibur

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Pollen contaminated frozen sections???
« Reply #4 on: August 09, 2006, 09:10:57 AM »
If an open window is near the cryostat, pollen could have contaminated the OCT or whatever embedding medium you are using for the frozens. If the pollen is in the OCT, it would stick to the slide just like the brain tissue when cut and not wash off. Is a nice flower garden under the window?  :D
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Erebus

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Pollen contaminated frozen sections???
« Reply #5 on: August 09, 2006, 09:36:30 AM »
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pollen could have contaminated the OCT or whatever embedding medium you are using for the frozens


I do not use any OCT, i mount the brains and slice them up for fixation in formaldehyde. The formaldehyde is far from the window.

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Is a nice flower garden under the window?


A parking lot, surrounded by suspicious-looking birches...

Offline excalibur

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Pollen contaminated frozen sections???
« Reply #6 on: August 09, 2006, 10:00:48 AM »
Ok, but after fixation, what is your procedure to cut the frozen sections?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Erebus

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Pollen contaminated frozen sections???
« Reply #7 on: August 09, 2006, 10:23:35 AM »
The brains are snap frozen -> cut in cryotome -> sections fixed in formaldehyde.

Offline excalibur

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Pollen contaminated frozen sections???
« Reply #8 on: August 09, 2006, 10:52:29 AM »
Oh Ok, do you do any cryoprotection before snap freezing?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Erebus

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Pollen contaminated frozen sections???
« Reply #9 on: August 09, 2006, 11:00:12 AM »
Nope, no cryoprotection.

Offline excalibur

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Pollen contaminated frozen sections???
« Reply #10 on: August 09, 2006, 11:10:19 AM »
Well, all I can think of then is you are using charged slides and the pollen is contaminating them at the cryostat by the window. Don't leave the box of slides open too long or the lid to the cryostat.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline discman

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Pollen contaminated frozen sections???
« Reply #11 on: August 11, 2006, 10:25:41 AM »
sorry, for jumping in here, but first thing I would do is close the window... 8)

I'm always wondering when I hear that people do labwork with open windows, but that's just me... ;)

GŁnther

Offline ImmunoNYC

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Pollen contaminated frozen sections???
« Reply #12 on: August 13, 2006, 01:48:25 PM »
Amazing! I learn something new every day.[/color]

Pollen contaminated frozen sections???
« Reply #12 on: August 13, 2006, 01:48:25 PM »