If anyone has experience with what to do or detail protocol from harvesting the colon from mice after sacrifice to preparing the tissue section slides and staining?
if any references please recommend
Thank you
Regards
KR
Dear Naesing,
it would be easier, quicker and probably cheaper to ask a histopathology lab to prepare your sections and also do the H&E and immunohistochemistry!
But if you insist on doing it on your own I am giving you some protocols:
1. Tissue fixation: When the samples are removed they should be adequately cleaned in H2O (if necessary) and placed immediately in 10% neutral buffered formalin solution for a period of more than 24 hours but less than 72 hours (depending on the thickness).
Formalin volume must be 15-20 times greater than that of your sample for sufficient fixation.
Formalin penetrates the tissue at a rate of 1mm/hour so you can do your calculations.
Do not exceed the 72 hour limit or you might have problems with immunohistochemistry.
(optional: you can place your samples in histology cassettes for easier handling. This can be done before fixation if the step depending on the size of your samples).
Helpful Link: https://www.youtube.com/watch?v=FVz6Ro0OUAE)
2. Tissue processing: After fixation samples have to be dehydrated by ascending alcohol mixtures, cleared by xylene and impregnated with paraffin.
I adjusted the protocol for samples larger than 4mm
Link: https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/
Dehydration:
1. 70% ethanol 30 min
2. 90% ethanol 30 min
3. 100% ethanol 30 min
4. 100% ethanol 30 min
5. 100% ethanol 1 hour
6. 100% ethanol 2 hours
Clearing:
1. xylene 1 hour
2. xylene 2 hours
Wax Impregnation: (Done at 57-60 °C)
1. wax 1 hour
2. wax 2 hours
3. Tissue embedding: After processing the samples are embedded in paraffin molds to produce paraffin blocks
Link: https://www.youtube.com/watch?v=xIyXA3c3oxU&t=5s
4. Tissue sectioning: This is the most challenging part!!!
Cutting the samples in sections of 3μm thickness (Must be done by experienced personnel) (For each sample cut slices and mount them on slides. Create as many slides as you need for H&E and for Immunohistochemistry)
After slide preparation incubate the slides in a chamber for 1 hour at 60 °C (not more) in order for the sections to adhere properly to the slides!!!
Link: https://www.youtube.com/watch?v=XDoTLJ3ZXtY
5. H&E staining: Follow the steps presented at the following link
https://www.youtube.com/watch?v=2D0rj0m6dVs&t=150s
You can substitute PBS with dH2O or even tap water.
You don't need to wipe the excess liquids...
You can use Gill's Hematoxylin, Harris Hematoxylin or Mayer hematoxylin, but you need to adjust the protocols for each type of hematoxylin.
6. Immunohistochemistry: For immunohistochemistry the slides need to be deparaffinized and then follows an epitope unmasking step. (For the second step you will need special kits that can be provided by the Ab providers)
It is imperative that your sections do not dry-out during the whole process!!!!
You can find an example at the following link: https://www.rndsystems.com/resources/protocols/protocol-preparation-and-chromogenic-ihc-staining-paraffin-embedded-tissue
I hope that was helpful
Kind regards
There are several methodologies depending on the specifications of your project but I believe ELISA immunoassay is the gold standard for cytokine detection and quantification. I don't think you will find any cheaper solutions for this task.
You can find several protocols at the following links:
https://www.bdbiosciences.com/en-eu/resources/protocols/cytokine-elisa
https://www.rndsystems.com/products/cytokine-elisa-kits
(and there are many more protocols at Thermofisher and Abcam....)
For intracellular cytokine detection you could utilize Immunofluorescence Flow Cytometry in order to detect the cell populations that express them.
Finally, one more option could be cytokine mRNA quantification for comparative analysis (and not at the protein level) but it is not widely used for that purpose. I also don't think this method is cheaper and the information you get is limited...
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