Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Our method makes mechanistic subcellular investigations possible in the fast-moving field of mechanobiology by fixing temporary protein modifications in place and then detecting them with specific antibodies. This method is technically easy to apply. The results are highly valid and reproducible.

Also, in comparison to other techniques, our method could really be used effectively in the emerging field of post-translational red blood cell signaling, which is dependent on temporary protein modifications, especially red cell mechanosignaling. To perform RBC protein fixation, dilute whole blood at a ratio of one to two in 4%paraformaldehyde solution for 20 minutes at room temperature. Centrifuge the blood solution at 132 g for three minutes at room temperature, and carefully remove the supernatant.

Resuspend the RBC pellet in two volumes of 0.1 molar PBS, and incubate for five minutes at room temperature. Again, centrifuge the sample, and remove the supernatant before resuspending the RBC pellet in one volume of 0.1 molar PBS. Label a microscope slide with the sample ID and the antibody applied using an alcohol resistant pen.

Add 10 microliters of the prepared RBC solution. Place a second slide on the sample at an angle of 45 degrees and disperse the sample evenly along the slide. Heat fix the slide by hovering the sample over the Bunsen burner with a constant movement for five to seven seconds.

For immunohistochemistry staining, mark the 2/3 area of the slide as a test and 1/3 as a control using a grease pencil. Then wash the sample areas twice by applying TBS for 30 seconds. After the second wash, add 0.1%trypsin, and cover the slide using a purpose-built incubation chamber with a lid.

Incubate the sample for 30 minutes at 37 degrees Celsius. Fill a beaker with tap water and stop the enzyme reaction by adding the tap water from the beaker onto the slide. Pour off the solution, and wash both areas three times with TBS.

After the final wash, add peroxidase blocking solution, and incubate for 30 minutes at room temperature. Following incubation, pour off the solution and wash both areas three times with TBS. Then add 3%skim milk solution and incubate for 30 minutes.

After incubation, pour off the solution, and add the primary antibody solution to the test area. Add antibody control solution to the control area. Incubate the samples at four degrees Celsius overnight.

The next day, pour off the antibody solution and wash both areas three times with TBS. Then add 3%normal goat serum solution to prevent unspecific binding and incubate for 30 minutes. Following incubation, add a secondary antibody solution and incubate for 30 minutes.

Pour off the antibody solution and wash the areas three times with TBS before proceeding to the development of staining. Next, to carry out an avidin-coupled horseradish peroxidase reaction, add diluted avidin peroxidase solution and incubate for 30 minutes. Place the slide under a microscope at 200 times magnification, and add freshly prepared DAB solution to both areas.

Monitor the staining of the RBCs continuously, and stop the staining by removing the DAB solution with a disposable pipette before the background starts to color. To perform sample dehydration, place the slide in a glass rack and immerse for five seconds each in increasing ethanol concentrations, and finally, in xylol. After dehydration, place the slide on tissue paper with the RBCs at the top to absorb excess liquid.

Add two, or three drops of mounting medium on a cover slip and cover the slide using the cover slip. For visualization and imaging, place the slide in a transmitted light microscope coupled with a camera. Turn on the light source and camera and start the software.

Under Bright-Field Mode, focus the RBCs by turning the coarse and fine focus adjustment knobs. Similarly, focus the RBCs under Fluorescent Mode. For analyzing RBC gray value, mark the edge of each RBC using the Oval Selection Tool within the ImageJ software.

Using the Measure command, measure the gray values of 50 RBCs from the test and 10 RBCs from the control. For immunofluorescent staining of RBC proteins, incubate the sample with a secondary fluorescently conjugated antibody for 30 minutes at room temperature, protecting it from light. After incubation, pour off the antibody solution and wash the slide three times with TBS.

Leave the TBS on the sample after the final wash. Dehydrate the sample and prepare the slide with a cover slip for microscopic evaluation as demonstrated. Turn on the microscope and fluorescent light source, and focus the RBCs under a Bright-Field Mode.

Capture bright-field and fluorescent images in at least three distinct areas of the test area of the slide selected at random. Set the exposure time to one second for fluorescent images and capture an image. Then switch to Bright-Field Mode, set the exposure time to Auto, and capture the corresponding bright-field image.

Save the images in TIFF format. Capture fluorescent and bright-field images in at least two distinct randomly selected areas of the control area of the slide. Save the images in TIFF format to preserve the original gray values of pixels, prevent compression, and carry metadata of the acquisition.

To measure the gray values of captured RBCs, open ImageJ. Mark each cell with the Oval Selection Tool, and analyze using the Measure command. Highlight between three and five areas free of RBCs and determine the gray values to measure the background signal.

To perform alternative data analysis of captured images, create a macro command through the Fiji release of ImageJ for automated selection, background correction, and gray value analysis of a given image. Open the TIFF format image for analysis in Fiji. Then open the RBC fluorescence.

ijm macro, and click Run. The signal of antibodies targeted against RBC Nitric Oxide Synthase, or NOS, phosphorylated at the serine 1177 residue at rest and in response to mechanical force exposure was assessed using both immunohistochemistry and immunofluorescence. A threefold increase in the signal of antibodies targeted against RBC-NOS phosphorylated at the serine 1177 residue was detected in response to mechanical force exposure of RBCs compared to the respective un-sheared cells when assessed with either the immunohistochemical, or immunofluorescence method.

The user needs to make sure that the antibody is only applied to the test area. Otherwise, the sample cannot be analyzed, because of a missing control. Given the availability of a wide variety of antibodies, targeting many different proteins and signaling processes is possible using this method.