This method can help answer key questions in the pathological and the immunohistochemistry. The main advantage of this technique is that faster enough and that produces digital media with high quality. Begin by fixing a three-by-three centimeter kung tissue sample in 4%paraformaldehyde for 24 hours at room temperature.
The next day, dehydrate the tissue in ascending ethanol immersions at room temperature, followed by three immersions in 100%xylene for 20 minutes per treatment. After the last xylene immersion, place the tissue in 63-degree Celsius paraffin for 30 minutes, followed by a second 45-minute immersion in fresh 63-degree Celsius paraffin, then change the paraffin one more time for a final one-hour incubation. When the paraffin has cooled, use a rotary microtome to acquire five micrometer-thick sections of the paraffin-embedded tissue sample, capturing the sections on 20 microgram-per-milliliter poly-L-lysine-coated slides.
For dewaxing, place the slides in a 65-degree Celsius oven for two hours, followed by immersion in dimethylbenzene for 15 minutes and soaking in 100%xylene for 15 minutes, then rehydrating the slides in a descending ethanol immersion series. For antigen repair, place the slides in an antigen-repair box and add 300 milliliters of citric acid repair liquid to the box. Heat the box in a microwave oven at high power for two minutes, followed by heating at low power to sustain boiling for six minutes.
When the slides have cooled to room temperature, wash the tissue samples with three five-minute 0.01 molar PBS washes in a rotary shaker at room temperature. To eliminate the endogenous peroxidase, incubate the slides with 3%hydrogen peroxide in double-distilled water for 30 minutes in a wet box at room temperature. At the end of the incubation, wash the slides as demonstrated and plug any nonspecific binding on each section with 100 microliters of 10%goat serum for 39 minutes at 37 degrees Celsius.
Next, label the tissues with 100 microliters of anti-ZWINT antibody per slide at four degrees Celsius overnight, followed by three washes in 0.01 molar PBS. After the last wash, incubate the slides with an appropriate secondary antibody for 30 minutes at 37 degrees Celsius, followed by three 0.01 molar PBS washes. After the last wash, add the slides to 300 microliters of fresh DAB for 10 minutes, followed by a 10-second wash with tap water at room temperature.
Counters stain the tissues with hematoxylin staining solution for two minutes at room temperature, followed by washing in tap water for 15 minutes, then dehydrate the tissue again in ascending ethanol immersions, followed by two 15-minute immersions in xylene at room temperature. After the last 100%ethanol immersion, mount the sections with 15 microliters of a 5%xylene in neutral gum solution and cover each tissue sample with a cover glass. For automatic whole-slide scanning of the sections, first allow the slides to dry at room temperature for 12 hours to desiccate the tissue samples.
Next, in the automatic whole-slide scanner, select Brightfield manually and enter the manual scanning interface. Revise the name of the slides and select a storage path for the images. Set the scanning area and select the scan samples using User-Set Thresholds option.
The threshold is about 50 with a scanning-area expansion value of 200 micrometers. Set the scanning focus value and select Automatic Focus and Single Layer mode, then load the slides onto the work station and start the automatic scanning. When all of the slides have been scanned, save the images for later analysis.
To analyze the images of the lung tissue sections, open the histopathological image-analysis software and select Local Computer to open the file with the scanning data. Select the appropriate zoom level and use Toggle Color Adjust to set the color to the optimal contrast, then circle and name the regions of interest on each image. Next, under plugins, select QC.The Scenario Builder will pop up.
Select Density Quant and adjust the detection bar. Adjust the score levels until the staining intensity of the circled areas is similar to that of the original images, and store and name the model as a new file, then apply the model to other slides and select Selected Annotation to let the software automatically calculate the APE score for the regions of interest in each image. Whole-slide digital scanning of lung tissue sections of tumor and adjacent non-tumor tissues from non-small cell lung cancer specimens produces digital images of a high quality.
APE scores calculated from these types of lung cancer samples are typically significantly higher than those determined for the adjacent non-cancer tissues. In addition, ZWINT expression levels in squamous-cell carcinoma tissue sections are frequently determined to be significantly higher than those measured in abnocarcinoma tissue samples. While attempting this procedure, it's important to remember to prepare with high quality of the tumor or tumor areas during making pathological scanning.
Following this procedure, other methods of the microscopy can be performed in order to answer additional questions. Neither the or different method for pathological scanning. After this technique paved the way for researchers in the field of pathological scanning to explore digital diagnostics in the research purpose.
Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as masks should always be taken while performing this procedure.
