Hello everyone,
My name is Kyana and I am a master' student in Animal Reproductive Physiology. I am trying to isolate RNA from chicken oviductal tissue and I am having some problems with my slides drying out faster than they should be in the LCM. I'm using RNA-free everything, I made the Mayer's and Eosin Y RNA free as well, and I'd like to show my protocol for staining. Any tips? I section my slides in the cryostat at OT: -22 CT: -18 at ~5-8 microns. If anyone has any tips on getting consistent tissue thickness slides that would be helpful too. Once a slide goes through this protocol, I wipe the excess xylene off and slip it into the Acturus Veritas LCM. Thank you for reading and responding!
Thaw: 5-7min depending on OCT thickness (all in coplin jars and the dipper is dried as much as possible in between jars)
95% etoh fixative: 1min
DI H20 agitated 2min
Mayer's H: 10min
DI h20: 1min
95% etoh: 1min
Eosin Y: 2 dips
95% etoh: 2min
95% etoh: 2min
100% etoh:
100% etoh:
100% etoh:
xylene 1: 2min
xylene 2: 2min
Based on the protocol you described, your slides should be completely 'dry' by the time they reach the LCM due to the ethanol/xylenes dehydrations.
Are you having issues recovering tissue from the slide?
Kindly go through the below link and find pdf files related to your question.
http://www.excilone.com/client/document/protocol-2_frozen-tissue-prep-and-staining_360.pdf
John Hardy Lockhart Hello and sorry for the late response. I am having trouble recovering RNA from the slide. Somedays isolation of cells work and other days it doesn't. I feel like the slides dry up too fast for good LCM targeting. Any suggestions?
How are you performing the captures? With the IR laser only, UV laser only, or both lasers?
What scale are you trying to capture? Single cells or regions of tissue?
John Hardy Lockhart Hello,
I learned to use both lasers since the UV melts the membrane to pick up the IR laser's cut material. I noticed that my samples only become dry and distorted (color turns to black or darker) when I start to place the cap at the region of interest to start laser calibration and then isolating cells.
Mehrajuddin Naikoo Hello, thank you for this protocol. I found some things I could change/alter with my own so I'll be trying out some tomorrow!
If you are capturing regions of > 100,000 µm2 you can pretty easily pull them off the slide using fine forceps and a dissecting microscope after cutting the membrane with the UV laser. Perhaps your tissue is adhering to the membrane more tightly than to itself, which should appear as only the regions under the IR spots being removed.
John Hardy Lockhart okay but what would you suggest if samples seem to dry out from xylene to the LCM before isolation? I'm talking about drying completely in minutes compared to the hours it should be/ used to be. I cleaned and made new media for everything. Is it the thickness of my samples? Or something else? They go from the correct histology color to dark purple/brown/black.
The purpose of the xylene bath a the end of the H&E staining is to dehydrate the samples and it should evaporate quickly. I work with unstained sections, and if the tissue is not completely dry it is very difficult to see. If you're no longer able to see the color of your stains after the xylene evaporates perhaps you need to increase the exposure time of the LCM camera. I see the tissue darken as it dries when using a low exposure time for my LCM sessions, going from translucent to black (see attached image).
You could try putting the samples back in xylenes, but I wouldn't recommend doing that. The liquid will get under the membrane and could interfere with visualization and/or capturing.
Hello,
Okay, I'll try increasing the exposure time. Since I am new to this, would you recommend a length of time for me to set it at? Also, I just read on the Arcturus Veritas LCM guide that xylene can "make the tissue very brittle and reduce the adhesion of the section to the PEN membrane". It recommends to omit the final xylene step but I have two. Have you had any issues with xylene making your samples brittle before?
I don't recall what exposure I typically use, but you can play around with that during your LCM session. It won't affect your tissue or captures, just how the tissue appears on the camera.
Xylene can make the tissue brittle/flakey, but I've only had issues when the tissue is partially off the PEN membrane (you can see some examples at the top of the image in my previous reply). I don't think it should be a problem unless you cut extensive regions and use insufficient IR spots on the cap.
Mehrajuddin Naikoo Hello,
I checked the manual and I noticed there isn't too much info on how to focus the beam if its unfocused and you can't focus the beam with the controls given. Any ideas on what to do?
John Hardy Lockhart Hello, I found the exposure and messed with the shutter speed and it seemed to help. My only other issue now, is that the capture laser's beam won't focus even with any manipulation of the coarse and fine focus tools being used. Any ideas what to do?
Kyana L Miller ,
Glad to hear that changing the exposure helped. There shouldn't be any need to focus the laser. It may be out of alignment with the camera, which can be fixed using the "UV Locate" and "IR Locate" tools (in Arcturus XT they are located within the "i" button of the microdissect panel at the bottom right of the screen).
So far, the director of our department and lab/machine manager are using the machine to see if the focusing laser will focus and it will not. The auto-focusing of the laser also asks us to manually find the laser at 10-20x ("The laser is not found will appear in red"). Is there a way to force or ask the capture laser to only automatically find the capture laser?
I'm afraid I don't have any experience with that system; I'm using an Arcturus XT. If they are unable to figure it out, I'm sure that someone at Arcturus can help.
There are some instructions in the Veritas manual though (see pages 52 - 53 of the attached PDF).
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