Histology

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Routine Histology

TACMASR offers services for making paraffin blocks from formalin fixed tissues and cells; snap freezing; sectioning paraffin and frozen material; and staining of human or research animal tissues, tumors, xenografts, bones and cell lines.

Please Contact Us if you have any questions or if you would like our specific SOPs (Standard Operating Procedure) for fixation, snap freezing or harvesting cultured cells.

Preparation of Tissue

Preparation of paraffin blocks:

Generally, tissues are harvested by the investigator and fixed in 10% neutral buffered formalin for approximately 24 hours, then transferred to 70% ethanol for up to 7 days. Fixed specimens in alcohol may be submitted to TACMASR for processing and embedding into formalin-fixed, paraffin-embedded (FFPE) blocks.

We generally batch process and embed weekly, so our turnaround time is approximately one week.

Optimal tissue size: Harvested tissues should be approximately 3mm thick x 15mm wide for optimal fixation. They should be no larger than 5mm thick, and no larger than 20mm wide – think of one or two stacked nickels.

Fixation: Immediately after harvest, place in 10% neutral buffered formalin for approximately 24 hours at room temperature. Transfer to 70% ethanol and store at 4C for no more than 7 days. Submit fixed tissue samples to TACMASR as soon as possible after transferring to alcohol.

Orientation:  Please talk to the TACMASR staff if you have specific or special instructions to orient and cut your specimens as needed.

Note: If at all possible, do not leave tissue specimens in either formalin or alcohol  longer than the recommended times!

Overfixed tissues are difficult, though not impossible, to cut. While histological stains may not be compromised with overfixation, immunohistochemistry may be.

Long term preservation of epitope specificity is antigen and protein dependent. Immunohistochemistry may or may not be compromised, therefore overfixation does not preclude an attempt at molecular analysis with immunohistochemistry (IHC).

IHC may or may not be compromised on thin sections cut onto microscope slides that have been stored for long periods of time at ambient temperature. Again, optimal preservation of specific antigens is protein dependent and does not preclude analysis.

Long term antigen preservation in the FFPE block is safest, with plans to cut sections within a month or less of IHC staining. Cut sections may be stored at -20C or -70C if at all possible.

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Snap freezing of tissue

Prior arrangements may be made for TACMASR staff to snap freeze freshly harvested experimental mouse or human tissue.

Optimal tissue size:  Freshly harvested tissues for snap freezing should be no larger than 3mm deep by 10 mm in size – think of three stacked dimes.

Snap freezing protocol:   Snap freeze tissue in a cryoboat surrounded by OCT. Immerse boat in isopentane cooled in liquid nitrogen. Transport snap frozen tissue blocks on dry ice until blocks can be stored permanently in a -80C freezer. Transport frozen tissue blocks to TACMASR on dry ice for cryostat sectioning. Contact us for a more detailed SOP.

Research Animal Tissue (mouse):

Fresh organs and tissues harvested by the investigator can be snap frozen or fixed in formalin for downstream histological, immunostaining or molecular analysis techniques.

Experimental mouse organs, tissues and xenografts generated by the UACC Experimental Mouse Shared Resource (EMSR) can be processed for histological techniques by TACMASR. If you are working with EMSR, please request that they hand off formalin fixed mouse tissues, bones and xenographs to TACMASR for processing/embedding and other services as indicated. 

Experimental mouse bones are decalcified in conjunction with formalin fixation and processing/embedding in order to soften the bones for subsequent sectioning. Histological and immunohistochemistry can be performed on decalcified bone tissue FFPE blocks. TACMASR decals bones in SurgiPath Decal I and II solutions.

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Cultured Cells

Cultured cell lines can also be preserved as formalin-fixed, paraffin-embedded (FFPE) blocks for histological staining and immunostaining. For example, treated vs. untreated cells can often be used as positive or negative controls for immunohistochemistry.

Making an FFPE block from cultured cells

Researchers grow their own cells and submit a pellet to TACMASR to make an FFPE block.

Generally, 1x106 cells from a T75 flask will make a nice cell pellet FFPE block.

Please arrange with TACMASR prior to harvesting cells, and plan to submit Monday through Thursday so that we can fix them in formalin overnight and prepare the cells for blocking the next day.

Harvest cells for making an FFPE block: Lift cultured cells in whatever manner you choose. Rinse in PBS in a 15ml conical tube, leaving a small amount of PBS on the pelleted cells. Transport cell pellet to the TACMASR lab on wet ice. TACMASR will fix cells in 10% neutral buffered formalin and prepare cells in a paraffin block, which can then be analyzed similar to an FFPE tissue block. Contact us for an SOP.

Cultured cells grown on coverslips for immunostaining

Researchers may culture cells on 12 mm glass coverslips* in whatever manner they choose, ie, different treatment conditions, and submit to TACMASR for immunofluorescence staining (IF).
(*TACMASS will supply the coverslips if you don’t have this size.)

Please arrange with TACMASR prior to setting up an IF staining experiment.

Culture cells for IF staining: Plate cells as usual into a 6-well plate or a 10mm or 30mm Petri dish (coverslips are hard to remove from a well in a 24-well plate). Use one or more wells per culturing or treatment condition. Use one coverslip per IF stain (ie, per antibody assay). Place up to three 12 mm glass coverslips in one 6-well, or numerous coverslips in a Petri dish. Incubate so that coverslips are not sitting on top of each other as the cells grow.

Grow cells to approximately 70-90% confluence.

Submit plates/dishes containing coverslips to TACMASR (with prior arrangement).

TACMASR will fix in methanol/acetone for optimal preservation of morphology.

Prior arrangements may be made with TACMASR to analyze IF stains.

Please Note: TACMASR does not accept:
  • fresh tissues without making prior arrangements with staff
  • cell pellets on Fridays without prior arrangements
  • any radiolabeled material

    As a courtesy, please include relevant information about potential biohazardous samples or conditions on the Request Form.

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Sectioning

TACMASR is equipped with a microtome to cut thin slices (sections) from FFPE blocks, and a cryostat to cut thin sections from snap frozen blocks. Sections are picked up onto microscope slides for histological staining or immunostaining.

Please Contact us if you have specific or special instructions to orient and cut your specimens as needed.

We routinely cut three micron sections from FFPE blocks or frozen blocks, and can cut thicker sections upon request (up to eight microns).

We can also provide investigators with unstained sections.

Investigators may request “step sections” - cutting through a block and collecting sections at (approximate) measured intervals.

We can cut thicker sections, up to 8-10 microns, and place them in directly into microtubes, from either FFPE or frozen blocks. Investigators can perform downstream applications such as protein techniques s(western blotting, immunoprecipitation) or molecular analytical techniques.

We accept FFPE blocks prepared in other histology labs for cutting/staining. Please be aware that the quality of sections cut greatly depends on the type and quality of the preserved tissue and block preparation. We can only be responsible for the quality of our own work.

Optimal slides for IHC: we have found that certain brands of glass microscope slides are incompatible with our IHC autostainer, eg, Surgipath X-tra® Slides.

The best slides for IHC staining on the Discovery XT Autostainer are VWR Brand Superfrost Plus microscope slides, catalog #43811-703. We have also used the equivalent Fisherbrand* Superfrost* Plus or ColorFrost* Plus Microscope Slides.

We also work with investigators to cut and stain both frozen and fixed sections for Laser Capture Microdissection (LCM).

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Histological Staining

Hematoxylin and Eosin staining (H&E) is a standard histological stain used by pathologists to recognize various tissue types and a broad range of cytoplasmic, nuclear, and extracellular matrix features. We routinely do H&E staining.

TACMASR can also perform certain special stains, including Trichrome, PAS, Giemsa and mucicarmine.  

 

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