Estudo do retículo endoplasmático e mitocôndrias Interações por

The overall goal of this experiment is to visualize and quantify endoplasmic reticulum mitochondria interactions using an in situ proximity ligation assay in fixed cells.This method can help to answer key questions in the biomedical field to better analyze ER mitochondria interactions in fixed cells using a simple immunofluorescence technique.Hopefully suggesting new treatment strategies.The main advantage of this technique is that we can visualize and quantify ER mitochondria interactions in fixed cells and this technique can be applied to paraffin embedded tissue biopsies of patients.Using the in situ proximity ligation assay demonstrated in this video, a mouse primary antibody directed against VDAC1 at the outer mitochondrial membrane and a rabbit primary antibody directed against IP3R1 at the endoplasmic reticulum membrane combine to their epitopes in proximity at the mitochondrial associated membrane interface.Proximity ligation probes that have DNA end tails attached and that are directed against mouse and rabbit IgG are added and can form templates for the ligation of connector oligos and can be amplified and visualized using Texas Red Oligonucleotides.This can occur only if the two targeted proteins are at a distance below 40 nanometers.In agreement with the distance between both organelle membranes, at mitochondrial associated membrane interface.To fix and block cells for the in situ proximity ligation assay, plate human H7 cells at 150, 000 cells per uncoded 35 mm glass bottom dish.The next day, remove the culture medium and use 1 mL of PBS to wash the cells.Then, aspirate the buffer and add 1 mL of ten percent formaldehyde.Incubate the plates at room temperature with agitation for 10 minutes to fix the cells.To stop the reaction, add 1 mL of 1 M glycine and mix the plates by rotation.Remove the glycine solution and add 1 mL of 1XPBS.Then, briefly agitate the plates and aspirate the buffer.Next, add 1 mL of 100 mM glycine to the cells.Incubate the plates at room temperature with agitation for 15 minutes.After aspirating the solution, permeabilize the cells by adding 1 mL of 0.1%triton-X100 in PBS.And repeat the incubation.Aspirate the detergent and add 1 mL of PBS to wash the cells.After removing the buffer, add 40 microliters of blocking solution to each dish of cells and incubate the samples in a humid chamber at 37 degrees celsius for 30 minutes.Then tap the blocking solution off the dishes, trying to obtain equal residual volumes of buffer for each slide while not allowing the slides to dry.To carry out the proximity ligation assay, use PBS to dilute the primary antibodies.Vortex and add the solution to the dishes.Incubate the samples at 4