The overall goal of this protocol is to quantitively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and image J.This method can help answer key questions in the tumor microenvironment field, such as how these components look in patient samples and how we can determine physiologically relevant ratios of cells. The main advantage of this technique is that it is straightforward for analysis in patient samples. The implications of this technique extend toward elucidating the undercharacterized, complex glioblastoma tumor microenvironment.
Generally, individuals new to this method will struggle because timing for chromogenic development needs to be closely monitored to prevent overdevelopment and saturation of the tissue. Begin the protocol by performing the washes specified in the accompanying text protocol. Dilute tris-based high pH antigen unmasking solution and distilled water using the manufacturers recommendations.
Move on to heat mediated antigen retrieval by adding the diluted unmasking solution to a non-sealed microwaveable vessel. Place slides into the vessel. Then place the vessel slides into the microwave.
Next, boil the vessel for 20 minutes at high power. Monitor the liquid levels for evaporation and replenish the vessel with distilled water as necessary. Allow samples to cool in solution for one hour at room temperature.
Outline the tissue sample with a hydrophobic to minimize the volume of reagents necessary to cover the sample. Then, pipe in enough permeabilization solution, 100 to 200 microliters to cover the tissue sample. Remove and discard the solution and cover the tissue sample with permeabilization solution again.
Incubate the samples at room temperature with blocking solution for one hour. Then, incubate the samples overnight in four degrees Celsius with primary antibodies diluted in blocking solution. The next day, detect primary antibodies using a horseradish peroxidase polymer reagent corresponding with the primary antibody host animal.
Pipe in enough permeabilization solution to cover the tissue sample and incubate the sample for five minutes. Remove and discard solution and cover the tissue sample in permeabilization solution one more time for five minutes. Incubate the slides in 0.3%hydrogen peroxide and IX TBS for 15 minutes.
Develop the samples with a peroxidase diaminobenzidine or dab substrate for two to 10 minutes until the desired stain intensity is achieved. Counterstain the samples to identify cell nuclei, such as with hematoxylin following the manufacturers protocol. Dehydrate samples with 100%ethanol and xylene.
Finally, mount the samples permanently with mounting media. Two regions of interest within our tumor resections, the primary tumor Bulk and the Adjacent regions, primarily composed of healthy tissue with diffused invading cancer cells were identified by collaborating neuropathologist on hematoxylin and eosin stained patient samples. Chromogenic immunohistochemistry identified positive staining for astrocytes, microglia, and oligodendrocytes within each patient sample.
We performed staining with no primary antibody for a negative control. The manufacturer recommended and further dilutions before settling on the one to 200 dilution due to optimal positive staining of cell body and process with minimizing background for the anti ALDH 1l1 antibody. Using Image J, the percent coverage of OSP-1 positive oligodendrocyte staining was quantified to eventually compare differences in our regions of interest.
Once the appropriate threshold has been applied to the image, the percent coverage from the threshold can be measured and compared for multiple patients. Five adjacent and bulk regions of glioblastoma or GBM patient samples, along with healthy brain tissue were compared for oligodendrocyte-specific protein one or OSP1 percent coverage. Once mastered, the staining technique can be done in four hours on the first day and two hours the second day if it is performed properly.
While attempting this procedure, it's important to remember to closely monitor the tissue staining intensity and hydration.
