Hi, my name's Michelle Tang from the Epigenomic Medicine and Human Epigenetics Laboratories. Headed by Dr.Tom Carianne, an associate professor ASAM Holster at the Baker IDI Heart and Diabetes Institute. Today I'll be demonstrating a gamma H to a X immunofluorescence assay in vivo determined the amount of DNA damage in disease tissues.
H two A X is a histone variant, which represents approximately two to 10%of total Sahel histone H two A and becomes rapidly phosphorylated on serum 1 39 of mammalian cells. In response to DNA damage, the phosphorylated form of H two A X is also known as gamma H two X, and it's been established that there is a direct correlation between the number of double stranded breaks and gamma H two A X gamma H two aax is a sensitive molecular marker and it's been shown to be an early indicator of double stranded brake. Therefore, it is a useful technique to detect DNA damage.
Gamma H two A X can be visualized as nuclear foci at the sites of DNA damage under fluorescent microscopy. For the purpose of this experiment, I'll be standing mouse lung tissues acquired from Dr.Simon Royce at the Murdoch Children's Research Institute. I'll be investigating DNA damage in lung tissues from a mouse model of chronic allergic airway disease, which shows many of the pathological features of human asthma, including airway hyperresponsiveness, airway remodeling, and airway inflammation.
So let's start the experiment. We begin by throwing samples to minus 20 degrees Celsius by placing them into the cryostat for approximately 20 minutes. For optimal cutting temperature SROs, microscope slides are used so that tissue sections do not detach from the slides.
During immunofluorescent staining, OCT is used to attach the samples to the chuck, place the chuck onto the cutting block, ensuring that it is securely attached. Set the cryostat to cut five micrometer tissue sections carefully begin sectioning pulling the samples towards you with a paintbrush place the microscope slide above the sections should adhere to the glass surface. Allow sections to dry at room temperature for one hour to ensure all moisture is removed before proceeding.
Firstly, we fix the tissue sections using 2%paraldehyde prepared in PBS without calcium and magnesium for 20 minutes. Tip off excess paraldehyde for placing slides into a Copeland jar for two 15 minute washes in PBS to perme, the cell membrane samples are treated with minus 20 degrees Celsius, 70%ethanol for 20 minutes following the permeation step. Samples are washed three times in PBS for 10 minutes each.
Remove each site individually to prevent the tissue sections from drying out. Block excess PBS from the slide and then block sections with 8%BSA in PBS containing 0.5%between 20 and 0.1%Triton X 100, also known as P-B-S-T-T. These slides are allowed to incubate for one hour at room temperature after the incubations samples are washed with P-B-S-T-T five minutes.
PBS TT is used to facilitate antibody penetration into the nucleus. Slides are then blood and dry to remove excess P-B-S-T-T. A circle is drawn around the sections with a pap pen to reduce the amount of reagents used.
Primary antibody, an anti phospho gamma H tox, rapid polyclonal antibody was used. A one in 500 dilution was prepared in 1%BSA in P-B-S-T-T. These sections were placed in the humid chamber and allowed to incubate for two hours at room temperature.
Tip off primary antibody before performing two five minute washes in P-B-S-T-T to remove excess primary antibody. Remove the slides and blot off excess P-B-S-T-T. Press the floor 4 88 goat anti Reid IJJ antibody was used.
A one in 500 dilution of the secondary antibody prepared in 1%BSA in P-B-S-T-T was added to the samples. Sections are then incubated for one hour in a humid chamber, tip off the secondary antibody before performing three five minute washes. N-P-B-S-T-T to remove excess secondary antibody.
Cover the Copeland jar in aluminum foil to prevent exposures to light for the duration of each wash step. Remove the slides and blot off excess P-B-S-T-T. The addition of 0.5 milligrams per milliliter of RNAs A in PBS was used to reduce any cytoplasmic staining.
Samples are then incubated at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. Following the incubation RNAs A is tipped off and samples are briefly washed in P-B-S-T-T to prevent sections from drying out. Keep samples wrapped in the aluminum foil before the addition of mounting medium.
Remove one slide at a time and blot off excess P-B-S-T-T. Add vector shield mounting medium containing propidium iodide a red nuclear stain over each section. Then carefully lower a cover slip trying to prevent trapping any air bubbles but off any excess vector shield on an absorbent paper.
Then now polish the edges of the cover slip to seal and prevent samples from drying out. Finally, store samples in a dark place at four degrees Celsius until slides are required for microscopy. A zes LSM five 10 meta focal microscope was used to acquire digital images using a 63 times all immersion objective and argon and helium neon laser are used at a wavelength of 4 88 nanometers for Alexa floor 4 88 and 543 nanometers for propidium IOD dyed.
Each field consisted of eight to 15 optical Z series splices with a step size of 0.5 micrometers and a one-time zoom. Images are exported as individual TIF files and analyzed using metamorph Images acquired from the zes LSM five 10 meta focal microscope are open in the metamorph program for image analysis and first eye counting. Fluorescent images acquired as a Z series are de convoluted and stacked to produce a 2D image using maximum projection filters.
This was done to minimize the loss of less intense foci. The maximum projected images are transformed into a binary image and morphological filters such as top hat are applied to reduce the background and also improve the sensitivity of foci detection. Corresponding propidium iodide images loaded and nuclear regions are identified.
These regions are emphasized by manually drawing regions around the nuclei to count the number of foci per field. The regions are transferred to the top hat images and light threshold filters are applied. The threshold was chosen carefully by visual comparison in concordance with the foci and to avoid inclusions of any background within each field.
Integrated morpho analysis for IMA was used to count the foci. Alternatively, manual counts can be performed to ensure reliability of results. And that concludes the immunofluorescent staining protocol in vivo to determine the amount of DNA damage in disease tissues.
This technique is suitable for the detection of DNA damage caused by different stimuli. However, it'll be most suitable to evaluate responses to ionizing radiation and radio sensitivity. So that's it.
Thanks for watching.
