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Immunohistochemistry

Immunohistochemistry image 1Immunohistochemistry (IHC) at PHL provides investigators with highly specific, highly sensitive tools to detect protein expression in multitudes of tissue from several species. Using antibodies, minute concentrations of specific proteins can be visualized within the confines of the microscopic/macroscopic environment from which the protein naturally resides. Antibody mediated staining can be used to confirm protein expression, detect protein co-localization, isolate cell populations, elucidate tumor locality, functionality, and/or type, and otherwise present a “snap-shot” of the tissue/cells for a specific target of interest. With around 20 years of experience, the PHL IHC department provides reliable and consistent results while continually developing novel techniques to meet investigator needs.

IHC solutions at PHL:

  • Standardized techniques for several commonly used antibodies.
  • Investigator specific antibody development and consultation.
  • Can utilize tissue/cell lines from numerous sources including, but not limited to, mouse, rat, rabbit, monkey, human, fish, plant, etc…
  • Wide range of control tissue readily available from mouse and rat, and fixed in a variety of fixatives.
  • Access to various mouse strains (C57BL6, FVB, SCID, etc…) for specific controls needed by investigators.
  • Stain and develop protocols for tissue from many fixatives such as 10% NBF, 4% PFA, Zinc (formalin and formalin free), Ethanol, Methanol, *In Development* IHC optimized fixatives and microwave fixation.
  • Stain and develop protocols for fresh, frozen tissue (OCT embedded).
  • Permanent, enzyme based visualization in brown, red, blue, and purple chromophores.
  • Immunofluorescence using highly stable Alexa fluors (488, 546 and 594).
  • Single, double, and triple label staining.
  • Bench top and automated staining for developmental projects where flexibility is needed and automated staining for established protocols providing standardization and speed.
  • Highly trained and knowledgeable staff.
  • All protocols are archived for future reference.
  • Several levels of quality control including pathologist review.
  • Comprehensive online database with over 250 protocols publicly available.

Routine IHC techniques:

Immunohistochemistry image 2

Numerous, well established protocols, used on a routine basis, are available for several antibodies which can be readily stained and returned to the investigator in minimal time. Common targets include: T-cells, B-cells, microvessels, macrophages, and apoptotic cells. Whenever possible, automated staining is utilized to provide standardization within and across multiple projects. As new protocols are generated, and thoroughly tested, they are added to the routine protocol list for future investigator use.

  • Quick turnaround time.
  • Batch staining for large projects.
  • Standardization using the Bond Max autostainer.
  • Many routine antibodies are available at PHL for use at minimal cost to the investigator.
  •  click + or - Common techniques/kits
    • Avidin/Biotin Complex (ABC Elite Kit, Vector®) targeting many species.
    • ApopTag Kit (Chemicon International®, S7100)

Developmental IHC techniques:

Due to the complexity of antibody/protein interactions, many antibodies need to be developed to work properly for IHC techniques. An intensive process involving investigator, pathologist, and technician consultation/interaction, antibody research, and thorough testing conditions are employed to provide the highest quality results.

  • Initial and continual interaction with the investigator.
  • Antibody research involving archived record searches, online database searches, manufacturer consultation, and pathologist interpretation
  • Highly flexible with modifiable protocols to meet specific conditions.
  • Several kits, buffers, retrieval options, secondary antibodies, and tertiary reagents available to test most conceivable stain-dependent parameters.
  •  click + or - Developmental Kits:
    • LSAB Kit (DAKO, K0690)
    • Animal Research Kit (DAKO®, K3955)
    • Mouse on Mouse Kit (Vector®, PK-2200)
  •  click + or - Matrix staining:
    • Numerous test conditions stained in one panel.
    • Conditions are logically applied based on previous research.
    • Quicker turn around.
    • Results are readily analyzed more effectively than developing over several weeks.
    • Potentially more cost effective than testing several conditions over a longer period of time.
  • Double labeling using 2 distinct chromagens or fluorochromes when required to label unique antibodies/targets in one tissue section.
  • Complete disclosure of techniques utilized for publications.

Immunofluorescence:

Immunohistochemistry image 3Immunofluorescence (IFC) couples the specificity of antibodies with the sensitivity of fluorescent markers. Today’s fluorescences are highly stable offering long lasting, vibrant assays for the analysis of cell populations, cell viability, and protein localization. IFC is an essential tool for detecting co-localization of proteins.

  • Stable and highly sensitive markers for visualizing antibody-protein interactions.
  • Provides clean, background-free images relative to chromagen staining techniques.
  • Allows for highly effective and accurate post-staining image analysis.
  • Excels in double and triple label antibody staining.
  • Critical in accurately analyzing the co-localization of proteins.

Cell Harvesting:

Quite often cell lines are submitted for either positive/negative IHC/ISH controls or individual investigators needs. PHL receives the cells and embeds a cell pellet into either paraffin or frozen OCT medium. To submit cells, please contact Tammy Beachley or Donna Butcher at the histology lab or Dr. Larry Sternberg for ISH projects. It is important to schedule cell submittal for early in the week to ensure optimal fixation/processing to paraffin block.

Cell Harvest Protocol (Pathology/Histotechnology Laboratory (PHL))
Immediately process the cells upon arrival.
Cells can be stored in a sealed flask at 37 o C for up to two hours or stored at 4 o C in a sealed flask overnight with media.

The following protocol is used for preparation of formalin fixed, paraffin-embedded (FFPE) cell block from either adherent or non-adherent cells growing in culture. Cells should be recently passed. The protocol is suitable when starting with 0.5 x 10 6 – 1.0 x 10 8 cells.

    1A. Adherent Cells : should be harvested without enzymatic treatment . Passage the cells the day before harvest. Remove culture supernatant and discard. Gently rinse cell monolayer with warm serum free TC media (i.e. add 25 ml per 150 cm2 via side of flask, rock gently, remove and discard). Add 10 ml warm serum free TC media per T-150cm2. Dislodge cells using a sterile cell scraper (“rubber policeman”). Transfer cells into a sterile, 50 ml conical centrifuge. Gently pellet cells from the media (centrifuge at 1000x g) 5 minutes. Continue with step 2 below.

    1B. Non-adherent Cells : Gently pellet cells from the media (centrifuge at 1000x g) 5 minutes in sterile 50 ml (or 15 ml) conical centrifuge tubes. Re-suspend pellet gently into 25 ml cold serum free TC media. Gently pellet cells from the media (centrifuge at 1000x g) 5 minutes. Continue with step 2 below.

    • Remove all but 1 ml (approximately) of media from pellet. Re-suspend cells by flicking the tube. Transfer suspended cells (via a 2 ml pipette) into a clean 1.5 ml conical ependorff tube. Pellet, 30 seconds at 8K in ependorff micro centrifuge. Discard supernatant, leaving approximately 100-200 ul behind.
    • Loosen pellet be gently flicking the tube. Add 8 ul of Thrombin stock at 1 unit/ul water, (Sigma #T-7009). Store Thrombin stock in single (10uL) aliquots at -20 o C. Thaw immediately before addition to cells, mix briefly by gently flicking the tube then hold treated cells on ice 2-5 minutes.
    • Add 5 ul of Fibrinogen stock at 10 mg/ml water (Sigma #F-3879). Store fibronectin stock in single use aliquots at -20 o C. Thaw immediately before use. Mix by gently flicking the tube. Incubate 2-5 minutes at room temperature.
    • Pellet clotted cells briefly, 20 seconds at 10K in micro centrifuge. Discard supernatant. Add 1.0 ml of room temperature 10% neutral buffered formalin or freshly prepared 4% paraformaldehyde (in PBS). Fix at room temperature a minimum of 8 hours, up to a maximum of 16 hours.
    • Following fixation, pellet cells 30 seconds at 8K in ependorff micro centrifuge. Discard supernatant (fixative) being careful not to dislodge pellet (dispose of properly).
    • Add 1 ml 70% ETOH in nuclease free water. Hold at 4 o C until transfer back to PHL for paraffin processing (3 days maximum).

Murine Immunohistochemistry Database:

A comprehensive database of tested IHC procedures composed of a variety of fixative, retrieval, and antibody combinations. There are currently over 250 publicly available records that detail the reagents and methods used for each successful technique. In addition, approximately 5,000 non-public, archived records help aid the development of future techniques as they arise. The database is continually monitored and updated with new entries on a regular basis.

 

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