Well characterized positive and negative cell pellet controls with defined protein or transcript expression levels are essential for developing immunohistochemistry assays. This protocol describes a process for creating and processing formula fixed, paraffin-embedded cell pellet controls. Such controls can be used to characterize the binding specificity while developing a new immunohistochemistry assay.
IHC assays are critical to our discovery and biomarker development efforts across therapeutic areas, and developing validated controls are essential for developing these assays. It is essential to heat the hydroxyethyl agarose based gel to at least 40 degrees Celsius before adding it to the cell pellet. The gel must be well mixed with the cells before solidifying.
Begin the fixation of the 293T-cell pellet by adding 30 milliliters of 10%neutral buffered formalin to a three milliliter cell pellet to create a 10 to one fixative to cell ratio. Resuspend the cells by repeatedly inverting the tightly capped 50 milliliter tube, and allow them to settle overnight at room temperature. To improve the fixation, increase the surface to volume ratio by inverting the tube the next day and resuspending the cells.
After 24 hours of fixation, centrifuge the tube at 930 times G at five degree Celsius for 10 to 15 minutes. Ensure the cell pellet is visible, and remove the fixative by decanting or carefully aspirating it using a sterile transfer pipette. Add molten hydroxyethyl agarose based gel at 40 to 60 degrees Celsius to the pellet at a one to four gel to cell pellet ratio.
Use a clean five inch and two millimeter tip sterling probe with an eye rinsed with tap water to gently stir the content of the 50 milliliter conical tube, creating an even suspension of fixed cells in the molten gel at the bottom. After evenly suspending the fixed cells in the molten gel, cap the chronicle tube and place it on the wet ice for five to 10 minutes. Once the gel pellet gets solidified, carefully place a clean microspatula along the side of the tube, and gently leverage the pellet without piercing it.
Place the solidified pellet onto the biopsy paper. Using a clean microspatula, trim the cell pellet into four to five millimeter thick slices to fit into a 26 millimeter by 26 millimeter by five millimeter tissue cassette. Place individual gel pellet slices in the center of a piece of biopsy paper.
Fold the two opposite sides, and wrap the slice in paper before placing it in a 26 by 26 by five millimeter tissue cassette. Close the lid. Place the trimmed cell pellet cassette into the tissue processor retort filled with 10%neutral buffered formalin, and run on a short processing schedule.
For embedding the cell pellet, place the processed cassettes into the holding area of the embedding center. Open the tissue cassette lid, and carefully unfold the biopsy paper. Place the cell pellet into a small 15 by 15 millimeter disposable embedding mold in cut side down.
While gently holding the cell pellet in the bottom of the mold with forceps, add 62 degrees Celsius tissue infiltration or embedding paraffin into the mold, covering the cell pellet. Move the mold to a cold block to solidify the paraffin. Adjust the cell pellet while the paraffin is solidifying, and fix it in the appropriate position at the bottom of the mold.
Remove the lid from the cassette. Place the cassette bottom side down on top of the embedding mold, and add extra molten paraffin to cover the cassette. When paraffin is filled over the tissue cassette, return the mold to the cold block for solidification.
Pick up the paraffin sections prepared using the rotary microtome from the flotation bath onto positively charged slides. Place the first section at the top of the slide within the cover slip and staining boundary, then serially place the next sections as one below the other. Once done, dry the slides initially at 23 degrees Celsius for 24 hours, then at 60 degrees Celsius for 30 minutes.
The embedded cell pellet prepared using this method showed an even cell distribution throughout the section, with minimal cell clumping and interactions in the pellet. When immunolabeling was visualized with the brown diaminobenzene, chromogen, and three cell lines varying levels of the TEAD transcription factor expression were observed in the nucleus, ranging from no, to weak, to the strong expression. Incorporating the cell pellets in a microarray allowed the evaluation of controls with varying expression levels in the same slide without variation in the procedures.
As shown in this example, negative control of PEG 10 deficient mouse embryonic stem cells and positive control of 293T-cells overexpressing PEG 10 can be seen. Ensuring cells are fixed adequately and distributed homogeneously throughout the gel pellet creates a uniform standardized assay control. The cell pellets can be used as controls in downstream immunohistochemistry and NC2 hybridization assays with standard antigen retrieval and labeling methods.
Cell pellet controls have enabled the development of many immunohistochemistry assays, particularly for novel or minimally characterized proteins. They serve as well defined controls that can help characterize antibody specificity.
