An initial meeting is necessary for researchers who are requesting histopathology services for the first time and or for a new project. Please contact histopathcore [at] email [dot] gwu [dot] edu (histopathcore[at]email[dot]gwu[dot]edu) or call 202-994-0036 to schedule a meeting. Please fill in and submit the tissue data Work Order Form here.
When submitting tissue samples for histology processing and evaluation, it is crucial to follow specific guidelines to ensure the quality and accuracy of the results.
Guideline for preparing Formalin-Fixed-Paraffin-Embedded (FFPE) tissue samples and cells for H&E, Special Stain (SS), Special Stain (SS), Immunohistochemistry (IHC) or Immunofluorescent (IF) staining
- At necropsy, isolate the tissue region of interest for each tissue that will be submitted for processing.
- Standard fixatives for tissue are 10% Neutral Buffered Formalin (10% NBF) or 4% PFA. Samples should be submerged in these fixative solutions with a volume to tissue ratio of at least 10:1, A standard fixation time is at least 24 hours at room temperature, but shorter fixation times (6-24 hrs) may be used for small tissue specimens. Tissue samples should be cut to a thickness of 3-5 mm to allow for proper fixative penetration.
Samples for immunohistochemistry (IHC) should kept in 10% NBF for no more than 48 hrs.
- After fixation is complete, most tissues can remain in 10% NBF if only routine processing and staining will be done. If tissues are small or immunostaining will be done on tissue sections, it is best to transfer the tissue to 70% ethanol for storage and transport. Tissues can be stored in 70% ethanol at 4 °C for a couple of months; however, to ensure optimal preservation and analytical quality, it is recommended that the tissue be processed within two weeks of fixation.
- We encourage submitting tissue samples in cassettes:
I. Label the cassettes with no more than 5-7 clearly legible characters, using only a #2 pencil, or a xylene- and alcohol-resistant marker.
(Test the labeled cassette with solvent to ensure the writing remains intact)
DO NOT LABEL THE CASSETTE WITH A STANDARD FELT-TIP MARKER.
II. Trim the tissue sample so that it fits comfortably within the cassette without being compressed or touching the cassette's outer edges when closed (2-3 mm thick).
III. Trimmed tissue should be placed into the cassette with the surface that is to be sectioned facing down.
IV.For minute tissue samples, use tissue sponges, biopsy bags, or biopsy wrap paper within the cassette to enfold or surround the tissue, protecting it and preventing it from slipping through the cassette openings. - Large tissue specimens may also be submitted in fixative to be grossed and trimmed by our histopathology core. If you would like the core to perform the trimming, please indicate the target structure(s) on your submission form and arrange to be present during the trimming process.
Guideline for preparing cell pellets using histogel for Paraffin Embedding
- Harvest cells and centrifuge to form a pellet.
- Carefully remove the supernant without disturbing the pellet.
- Resuspend the pellet in 10% neutral buffered formalin.
- Fix for 1-2 hours at room temperature or overnight at 4C.
- Recentrifuge to form a pellet and aspirate liquid without disturbing the cell pellet.
- Embed in histogel:
- Prepare the histogel by warming (to about 65C) the gel until it becomes liquid.
- Add the liquid histogel to the cell pellet and mix the cell pellet with the histogel.
- Allow histogel containing the cell pellet to solidify by cooling, using a cooling plate, ice cube or an ice pack
- Place the solidified histogel in biopsy wrap paper in a cassette.
- Secure samples by enfolding the histogel in the biopsy wrap paper. Close the cassette and submerge in 70% alcohol for storage and transport.
Guideline Frozen Tissues and Cryosections for H&E, Special Stain (SS), IHC or IF staining
Frozen Tissue Samples should be submitted using optimal methods for preserving tissue morphology and antigenicity for rapid analysis, especially in studies using immunohistochemistry. Immediate processing is needed after devascularization and necropsy to prevent autolysis and crystal artifacts.
- Blot tissue dry to remove excess fluid.
- Place tissue in legibly labeled cryomold with a small amount of OCT. Orient the tissue so that the surface to be sectioned is facing down.
- Add more OCT to fully cover the tissue. Ensure that there are no bubbles.
- Snap freeze the tissue sample by placing the cryomold in an isopentane bath within a liquid nitrogen vapor or on dry ice.
- When the OCT is solidified, store samples at -80C for later testing.
- Please submit your frozen tissue on dry ice to the core lab
For Fixed frozen Tissue:
- Rinse tissue samples in cold PBS
- Fix the tissue sample in 10% Neutral Buffered Formalin or 4% PFA , typically for 24 hours (or 6-24 hrs for small specimens) at room temperature.
- Transfer to 30% sucrose in PBS and store at 40C overnight. Tissue should be fully submerged in the solution.
- Samples should be embedded in OCT and frozen, as above, within the next 24 hrs.
Guideline for Submitting Slides for scanning to Whole Slide Images (WSI)
- Ensure that the coverslip fully covers the tissue section and that it is properly aligned on the slide and free of bubbles or excess mounting medium.
- Slides must be completely dry before scanning, moisture or sticky mounting medium can damage the scanner.
- The slide and coverslip should be free of fingerprints, dust, and dirt.
- Any pen markings on the slide should be removed, as the scanner will consider such marks as tissue and be unable to focus properly.
Please schedule a drop-off time by email (histopathcore [at] email [dot] gwu [dot] edu (histopathcore[at]email[dot]gwu[dot]edu)), or phone call (202-994-0036). Please wait for confirmation before dropping off your samples.
Please submit your service request by filling this form to the best of your ability.