Hi there, I am Raja Ti Doing my PhD at the Baker IDI Heart and Diabetes Institute. I'll be taking you through the steps that are involved in 3D illustration of Eastern modification that marked DNA damage and chromatic condensation in a new cardiac cell. DNA is wrapped around nuclear zones.
Each nucleosome is made up of four different types of histones, H two A, H two b, H three, and H four radi. The formation of DNA breaks and this is marked by PHO of H two X.This pho of H two X occurs mainly in creatine that is marked by methylation of H three K four. Now we'll be looking at the 3D illustration of modifications in your cell immuno label with the pho layer H two X and the methylated H Three K four.
Let's get started. We begin by placing E course of the cell suspension into 15 mil. Falcon tubes S are washed twice with PB scentation at one 50 G and Resus.
Suspend in fresh Media to see the immediate effect of Radiation on cellular events. Keep cells on eyes for five to 10 minutes prior to and during radiation. Expose cells to either gamma radiation or x-rays After radiation, dispense three milliliters of the cell suspension into each GU of six.
Well plate for peak gamma H two X incubate cells between 30 minutes to one hour at 37 degrees centigrade. We use the Cytosine mission to place the non-adherent cells onto glass slides. When assembling the cytosine clip, make sure that the whole filter cord coincides with the funnel.
Dispense one 50 microliter, swab this cell suspension Into each cytosine final place the cytosine clips into Cytosine mission and Spin it 500 RPM for five minutes. Remove the cytosine clips and separate the slides from the filter card. Do not let cells air dry too long as it may influence the Background in fluorescein staining.
Using liquid blocking pin draws circles Around each smear, we fix the cells with 4%paraform head at room temperature for five minutes. Compared to ethanol or ol, a better fixation was observed with 4%paraform head. Wind slides with PPS perme cells with Triton X 100 at room Temperature for five minutes.
Compared to twin 80, we observed a better signal in cells per with Triton X 100. Watch cells with PP S3 times each for five minutes. Block cells in one person BSA for 10 minutes at room temperature and repeat this three times when compared to other blocking agents and times.
BSA was most efficient In minimizing non-specific binding of primary antibodies, we have used two primary antibodies diluted one in 500 with 1%BSA at approximately 50 microliters of primary antibody to each slide. Incubation of primary antibody at room temperature for 60 minutes reduces background staining compared to overnight incubation at four degrees centigrade. Wash cells three times with PBS for five minutes.
Alexa floor 4 88, good anti mouse and Alexa floor 5 46 word anti rabbit. Secondary antibodies were used to stay different markers simultaneously. Use antibodies raised in different species.
Add approximately 50 microliters of diluted secondary antibody to each slide. Incubate cells at room temperature for 90 minutes. It's recommended to incubate cells in your moist chamber as it'll prevent cells and antibody from drying wash slides with PB S3 times each for five minutes.
50 microliters of a one in 500 dilution of topo three in PBS was added to each slide. Wash slides with PBS for three times each for five minutes. Remove excess moisture from slides and add Antifa solution.
It is recommended not to dry cells completely before adding Antifa solution. Place a cover slip onto the slide. K should be taken to avoid the formation of any air bubbles.
Leave slides overnight in a dark moist chamber. Seal the slide with nail polish to prevent The drying of cells. Images acquired an a confocal Microscope reveal.
Better spatial resolution are gone and helium laser were used for the image acquisition. A 63 oil emissions object two lenses preferred to higher or lower magnification lenses. A zoom factor of three or four will produce a better resolution image.
For FO accounting from many cells, it is preferable to use a scan speed of eight, an image size of 1024 into 1024 pixel cells. When imaging for multiple channels, a sequential line scan acquisition is recommended compared to frame scan. To avoid specimen bleaching, the detector gain and amplifier offset are adjusted to reduce the mice and signal saturation.
Since the size of Kama HX X four may be lower than 0.5 micrometers, it is recommended to acquire images with at least 0.5 micrometer. Step size along jet axis images are required in a jet series pattern using the mark first and mark lost pattern. To illustrate the spatial relationship between different proteins, which is recommended to use a scan speed of six with image size of 2048 And 2 48 pixels using metamor first.
Second can be counted From images acquired on various confocal microscopes. Open gamma hgx image tags either directly from file menu or by using bill numbers.Tags. Go to process menu and choose Stack arithmatic.
Choose maximum projection and select the plans to be included. And click okay. Now choose top hat and select Gamma H two X image as a source image apply.
Top hat filter and resulting image will be a binary image with less noise and reduced variation in foci intensity. Go to regions and select region drawing tool and draw regions around nuclei. Go to process, menu and sell threshold image.
Choose inclusion threshold and set the lower and a higher threshold values. It is usually the threshold values that affect the four number. The optimal threshold value is the one that minimizes the inclusion of background and maximize the foray inclusion.
This can be best achieved by counting foray number using different threshold values. And then compare foray number with naked eye counting. Select DPI image and go to regions menu and select transfer regions option.
Now we have corresponding foray and nuclear images ready for counting. Most of the nuclear regions are transferred to top hat. Go to measure, menu and select integrated ary analysis or IMA select Measure all regions and click on measure to obtain FTE number.
Now the FTE numbers from each region can be exported into spreadsheet by using Log data option. Open the previously Created jet projector images for each channel. Go to display and select color align.
Select the respective source images for each channel to create a image image for all three channels. Line scan analysis is performed to illustrate the spatial positioning of different markers in the nucleus. Go to measure tool bars command and select line scan analysis dry line across the cell.
This will create a graph showing the fluorescent intensity of each channel along the line and represents the distance of different markers With respect to each other. To create a 3D image from a stack, go To a stack menu and choose 3D reconstruction. Select 3D reconstruction type maximum.
Choose the plan of rotation, choose jet calibration distance. Usually the user specify click okay to create a 3D reconstruction. And that concludes our protocol for Immuno fluor and staining and image analysis.
In summary, immunofluorescence confocal microscope is two technique for the three dimensional visual of different C markers and gamma H two X foy in cell nucleus. Thanks for watching.
