Difference between revisions of "20.109(F20):M1D4"

Part 1: Complete staining for gamma-H2AX assay

1. Make sure to have TBS solution available before you start. Aspirate the secondary antibody solution off the coverslip and immediately add 150 μL of TBS. Do not let the coverslips dry out during this process.
2. To complete the post secondary wash, add 150 μL of TBS per coverslip, let incubate at room temperature for 3 min covered, then aspirate.
   • Repeat this step twice.
3. Obtain glass slides from the front laboratory bench and label your slides with all of your experimental information and group name, add one drop of mounting media to the slide.
4. Aspirate the final TBS wash and using tweezers place the coverslip cell-side down on the mounting media "spot" on the microscope slide. Try your best to avoid bubbles by slowly placing the coverslip over the mounting media.
   • The cell-side of the coverslide is the side that was facing up in the staining chamber.
5. Complete Steps #3-4 for coverslips from all of the coverslips you stained.
6. Add one small drop of nail polish to each side of your coverslip to seal it to the glass slide.
7. Alert the teaching faculty when all of your microscope slides are ready and you will be escorted to the microscope in the Engelward laboratory.

Part 2: Analyze γH2AX images

Please obtain the raw γH2AX images from the Class Dropbox folder. There are two folders--let's begin with the one named "Two channel images" and save the other one for the next section. Three sets of images (i.e. image stacks) were taken per condition, and each image stack contains images from two channels: DAPI (blue) and Texas Red (red). Remember that the secondary antibody used for the γH2AX staining was conjugated to an Alexa594 fluorophore, which emits red light. For each image stack, you will use ImageJ to 1) identify the location of the nuclei using the DAPI channel and 2) quantify the total γH2AX fluorescence in the Texas Red channel at locations specified by the DAPI channel.

Identify intensity thresholds for DAPI channel Example of thresholding cell nuclei using ImageJ

First, you will identify intensity thresholds that will properly identify the cell nuclei in all the images. To be consistent and fair in analyzing fluorescence images, it is good practice to use the same intensity thresholds on all the images.

   Open ImageJ.
   Open one image stack from the no treatment condition.
       The first image you see is the DAPI channel
       If you scroll to the right, the second image in the stack is the Texas Red (γH2AX) channel.
   While the image is on the DAPI channel, go to Image -> Adjust -> Threshold.
       A threshold window should pop up
       Check the box for "Dark Background"
       Make sure the cell nuclei are highlighted in red.
       Adjust the threshold values to properly identify the majority of the cells' nuclei.
       Record the threshold values.
   Repeat this process for one image from each condition and cell line, and settle on threshold values for the DAPI channel that you will then use to analyze all the images. Write these values in your notebook.
       It is best to define the lower threshold value based on your images, and set the upper threshold value as 256, which is the maximum possible intensity value for a 8-bit image.
       You can type in threshold values by clicking on the "Set" button in the Threshold window.
   Close all open images (File -> Close All).

Test γH2AX quantification on one representative image

   In ImageJ, open one image to test the FITC quantification protocol.
   Split the image stack into two separate images.
       Go to Image -> Stacks -> Stack to Images.
       The DAPI image will have "-0001" as a suffix in its title.
       The Texas Red (gamma-H2AX) image will have "-0002" as a suffix in its title.
   Duplicate the DAPI image and turn it into a mask to identify nuclei locations.
       Click on the DAPI image.
       Go to Image -> Duplicate, and click OK on the default title.
       Set the thresholds you chose on the duplicated DAPI image to identify nuclei.
           Go to Image -> Adjust -> Threshold.
           Check the box for "Dark Background".
           Click on the "Set" button and type in your threshold values (use 256 for the upper threshold level).
       Go to Process -> Binary -> Convert to Mask.
           This makes the image black and white, where the white areas should correspond to nuclei locations.
   Use the newly created mask to identify locations on the Texas Red channel in which to quantify the gamma-H2AX signal.
       Go to Analyze -> Set Measurements.
           In the Set Measurements window, make sure the following boxes are checked: Area, Mean gray value, Min & max gray value, Shape descriptors, Integrated density, Display label.
           In the "Redirect to" field, scroll and select the Texas Red image (suffix -0002). Then press OK.
               This will direct ImageJ to the Texas Red image to analyze the metrics you selected in the areas identified by your mask. This will give you information about the gamma-H2AX signal in each nucleus.
   Run the analysis by selecting Analyze -> Analyze Particles.
   Example of areas identified by intensity thresholds for DAPI channel in ImageJ
       In the "Size" field, type 200-Infinity. This will eliminate small, extraneous particles that do not correspond to nuclei.
       "Circularity" can remain at default values: 0-1.
       "Show" should say "Outlines".
       Click the following options: Display results, Exclude on edges, Summarize.
       Press OK to complete.
   A window will pop up showing outlines of each nucleus the software identified based on the thresholds you defined. Each identified area is labeled with a red number, corresponding to the left column of the data shown in the "Results" window.
   Take a look at the "Results" window to see the results of the analysis. It is good practice to validate the numerical results by comparing them to what you see in the images.
       The definition of the various measurements performed can be found on the ImageJ website (linked here).
       Does the nucleus with the largest "Area" correspond to the biggest nucleus you see in the drawing? The area here is in units of square pixels.
       The RawIntDens field is the total intensity (sum of the intensity of all the pixels) of the corresponding region. Does a region with a high total intensity value correspond to a cell with a high gamma-H2AX signal? Click on the Texas Red image to double check.
   Close the "Results" window and do not save the data, as you will run the analysis on all the files together next.
   Close all open windows in ImageJ (File -> Close All).

Quantify gamma-H2AX signal in all images

   Ensure that all of your H2AX images are in one folder.
   Download AnalyzeH2AX_FITCintensityBatch_Fa19 script (linked here).
       Right click on the link and download the file into a folder where you can find it.
   In ImageJ, go to Plugins--> Macros--> Run, and click on the AnalyzeH2AX_FITCintensityBatch_Fa18 script that you downloaded.
   When the script prompts you to "Choose input folder," choose the folder containing all your .tif image stacks (folder named "Two channel images"), and click "Open."
   In the dialog box titled "Choose Intensity Threshold Values," type in the corresponding DAPI threshold values you have chosen, and click "OK."
   You will be prompted to name the resulting Excel file next.
   Please wait for the script to run through all your images. In the end all the image files will pop up, along with the "drawings" that show where it identified cells in your images.
   The script will output one Excel file into your image folder.
   Before closing any images, validate the results in the Excel file with the images in ImageJ.
       Choose a few representative images to verify.
       Check the "Drawing" images and DAPI images to see if the nuclei were called correctly by your threshold values. For example, if two or more nuclei were counted as one region, throw out the data point on the Excel sheet. If the nuclei were consistently identified incorrectly, consider choosing new threshold values and repeating the analysis.
   When you are finished validating the data on the Excel sheet, post your data under the "Raw γH2AX Data" column on the Class data page then close all the image files (File -> Close All).

To share your analyzed data with the class, complete the Excel template (linked here), and post under the "Analyzed γH2AX Data" column on the Class data page.