[Histonet] Cryostat sections with small bubbles

[Alan Bright]

Dear Kathleen,
 
                        Thank you for your reply. I do understand that Specimen Temperature Control may not be available to all cryostat users. Therefore I will set out a method that I have posted on Histonet previously to assist with sectioning problems on brain. You will see that this is a far more scientific method than warming up the face of a specimen with ones finger and will render repeatable good quality sections.
 
Set the cryostat microtome chamber to -8n to -12ºC, place containers made from aluminium kitchen foil around and in contact with the knife in areas that with not impede the specimens travel or the anti-roll plate, also do the same for where the anti-roll plate is parked away from the knife. Fill these containers with graduals of Solid C02. Allow 10 minutes for the solid C02 to cool the knife/ anti-roll plate to a lower temperature than the specimen, then start sectioning. 
 
Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright <@t> brightinstruments.com
Web Site: www.brightinstruments.com

 

	-----Original Message-----
	From: Kathleen Spencer [mailto:kspencer <@t> utmem.edu] 
	Sent: 16 June 2005 16:05
	To: Alan Bright
	Cc: Emily Jane Wiesner-Camm; histonet <@t> lists.utsouthwestern.edu; Katri Tuomala
	Subject: Re: [Histonet] Cryostat sections with small bubbles
	
	
	Yes Alan, but when I put my finger on the block face to warm it, there is a kimwipe or gauze between my finger and the tissue. It works really well, so I guess the Ark is still around. 
	As the owner of a company that sells cryostats, you need to be aware that not everyone has or can obtain such devices. 
	Many of us, especially in research, work with old and crippled equipment, thus we need all the help we can get from our fellow technicians. 

	Kathleen Spencer, HT ASCP 
	Lab Manager/LCM Supervisor 
	UTHSC 



	On Jun 16, 2005, at 5:11 AM, Alan Bright wrote: 


		Emily, 

		Basically you need to section brain at -8 to -12ºC as colder temperatures will cause what you are getting plus cracking. However to achieve good quality sections at these temperatures you will need to maintain the knife and anti-roll temperatures at -20ºC or lower to allow the section to slide in between the knife and the anti-roll plate unimpeded. Which is what Kathleen is doing when she warms the block face with her finger, a technique that went out with the Ark and should not be used as firstly you could have an infection issue and secondly it is not a very scientific approach in this day and age when there are devices fitted to some cryostats such as independent specimen temperature control to perform this task with full and repeatable control and of most importance, ease. 

		Best Regards 

		Alan Bright 

		Bright Instrument Co.Ltd. 
		St Margaret's Way 
		Huntingdon 
		Cambridgeshire 
		PE29 6EU 
		England 

		Tel No:+44 (0)1480 454528 
		Fax No:+44 (0)1480 456031 
		Email: abright <@t> brightinstruments.com 
		Web Site: www.brightinstruments.com 



		-----Original Message----- 
		From: Kathleen Spencer [mailto:kspencer <@t> utmem.edu] 
		Sent: 15 June 2005 16:50 
		To: Katri Tuomala 
		Cc: histonet <@t> lists.utsouthwestern.edu; Emily Jane Wiesner-Camm 
		Subject: Re: [Histonet] Cryostat sections with small bubbles 


		Hi Emily, 

		I cut rat brain sections at -18 to -20 and often have to warm the block 
		face with my finger before each section. I also have better luck if I 
		cut sections at 10u. These are fresh frozen brains. If I cut them at 
		20u I get bubbles under the sections. I keep them in the cryostat, and 
		then fix the sections in cold fix, buffer wash, water wash, then air 
		dry them in the hood. 
		When I cut rat brain that has been perfused, I cut 20u floating 
		sections. They never adhere well to slides, even if I use a hot plate. 
		I always have bubbles under the section. That is why in this case we 
		use floating sections. 

		I hope this is helpful. 

		Kathleen Spencer 


		On Jun 14, 2005, at 8:39 PM, Katri Tuomala wrote: 


			Hi Emily, 
			I have never heard of hot plating cryostat sections. You may be 
			boiling the moisture under the section forming bubbles or do you air 
			dry them well first? Also -35 C seems too cold for brain sections, you 
			may be getting some cutting artifacts. 
			I'm sure there'll be others with helpful information for you. 
			Katri 

			Katri Tuomala 
			Hamilton, Ontario, Canada 
			----- Original Message ----- From: "Emily Jane Wiesner-Camm" 
			<Emily.Wiesner <@t> medecine.unige.ch> 
			To: <histonet <@t> lists.utsouthwestern.edu> 
			Sent: Tuesday, June 14, 2005 10:36 AM 
			Subject: [Histonet] Cryostat sections with small bubbles 



				Hi All, 
				I was wondering whether anyone can let me know why small bubbles 
				appear underneath my rat brain sections when I cut them on a cryostat 
				(at -35) when they appear perfectly flat before placing them on a 
				hotplate. How they can be avoided? 
				Thanks, 
				Emily 


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