The overall goal of this procedure is to visualize the location of viral RNA in C two within cells. To accomplish this first, grow the cells that will be transfected or infected on glass cover slips. The second step is to fix the cells with 4%paraform aldehyde after successful transfection or infection.
Next, incubate the cells with a hybridization mixture that labels the RNA. This is done by placing the cover slip cell side down on the hybridization mixture on a slide. The final step in this procedure is to use antibodies to stain the cells for the RNA label.
Ultimately, the localization of the labeled RNA within the cell is visualized by fluorescence in C two hybridization or fish. The main advantage of this technique over other methods like RNA imaging and life cells using tags is that native RNA can be detected as opposed to modified RNA. Generally, individuals new to this technique will struggle because it requires patients in care and handling cover slips, standardization of method, as well as precise timing.
During incubations, The specific procedure for culturing cells for fixation will depend on the type of cells. For adherent cells, it is initially important to seed them onto untreated sterile glass cover slips. So the final co fluency upon harvesting is approximately 70 to 80%Non adherent cells should be cultured the standard way until they're ready to be collected.
Then incubate them with cover slips that have been treated with poly L lysine for a typical 12 well polystyrene tissue culture plate plate 150, 000 cells per well at 24 hours prior to infection or transfection. Incubate the cells on the cover lips for one hour at 37 degrees Celsius to begin the procedure for cell fixation. Discard the media by careful aspiration and gently add one XPBS prepared and double distilled water.
Or DPBS wash the cells for one minute. Dathyl pyro CARBATE or DEPC treated one XPBS may also be used. Do not use untreated PBS as it may contain R nase enzymes.
Discard the DPBS and gently add enough. 4%para formaldehyde to cover the cells completely incubate for 15 to 20 minutes after that, discard the paraldehyde and wash the cells with DPBS for one minute. Discard the DPBS and add 0.1 molar glycine incubate for 10 minutes.
Next, discard the glycine and wash the cells with DPBS for one minute. After discarding the DPBS add 0.2%Triton X 100, incubate for five to 10 minutes. If staining for nucleolar or nuclear envelope proteins perme with triton X 100 for no more than five minutes or the protein localization will become diffuse.
Discard triton X 100 and wash two times in DPBS for one minute each time prior to beginning the procedure for fish. Prepare sides on which to incubate the cover Slips taking care to clean and label them well. For an 18 millimeter cover slip, add 50 microliters of DNA solution to the slide.
The most difficult aspect of this procedure is the proper handling of cover slips. It's important to make sure you incubate the right side of the cover slip and to handle them gently. To avoid breakage, Add the cover slip to the slide, being sure to place it cell side down and incubate for 15 minutes at room temperature After 15 minutes, place the cover slips sell side up in a culture dish with one X-D-P-B-S.
Wash the cover slips for one minute. This mix contains the following. 50%deionized form amide one milligram per milliliter, TRNA two X-S-S-P-E five X dnar RNAs out probe and DEPC water to a final volume of 50 microliters.
The probe is a dig genin labeled RNA probe designed to be complimentary to the viral genomic RNA.Place. Each cover slip cell side down on the hybridization mixture on the slide. The incubation should be performed in a tray containing a 50%FIDE two X-S-S-P-E.
Mix incubate for 16 to 18 hours at 42 degrees Celsius. Next incubate cover slip cell side down in 50 microliters of 50%fide for 50 minutes at 42 degrees Celsius. Wash the cover slips twice in two X-S-S-P-E by incubating themselves side down in 50 microliters of two X-S-S-P-E for five minutes each at 42 degrees Celsius.
Finally, wash the cover slips in one X-D-P-P-S for one minute. To begin this procedure, block the cover slips by placing the cell side down in 50 microliters of one X Roche blocking solution for 30 minutes at room temperature when blocking is complete, place each cover slip cell side down in 50 microliters of a primary antibody solution that contains all primary antibodies for the experiment at their appropriate concentrations. Incubate for one hour at 37 degrees celsius after an hour.
Place cover slip cell side up in a culture dish containing DPPS and wash cover slips for 10 minutes. Next, pipette 50 microliters of secondary antibody solution onto a slide for each cover slip. Alexa floor conjugated antibodies can be used to generate a range of colors and are used at a one to 500 dilution in one x blocking solution and one X-D-P-B-S Place each cover slip cell side down in the secondary antibody solution and incubate for one hour at 37 degrees.
Wash the cover slips twice for 10 minutes each. In DPBS dry the cover slips cell side up on a piece of blotting paper. Be sure to cover the cover slips to avoid exposure to light and bleaching.
Once all the liquid has evaporated, mount the cover slips onto fresh slides. Using eight microliters of immuno mount gently tap down the cover slip to eliminate any air bubbles apply nail polish to the edges of the cover slip to secure it in place. The visualization of RNA and proteins by microscopy techniques is also critical to the success of this procedure.
Settings on the microscope used can help or hinder visualization. Therefore, it is key that the settings on the microscope be set correctly and are consistent from one sample to another in a given experiment. A representative result of viral RNA visualization by fish and protein localization by immunofluorescence is illustrated by this figure.
In this first panel where hela cells are transfected with HIV one, the viral RNA for HIV one tracted in green is seen throughout the cytoplasm displayed diffusely for the most part, although small cytoplasmic punte are not uncommon, the specificity can be seen by comparing positive cells to surrounding cells that display no fluorescence as illustrated by the black and white inset image that represents only viral RNA staining. The red stain reflects immunofluorescence of Ross GA PSH three domain binding protein or G three bp, which primarily marks the cytoplasm when helo cells are transfected with HIV one that lacks the regulatory rev protein. The viral RNA produced is retained in the nucleus.
This can be visualized as a bright green signal in the nucleus and a lack of RNA fluorescent signal in the cytoplasm. The distribution of the HIV one viral RNA can also be altered by overexpression of certain cellular proteins. For example, the overexpression of RAB seven interacting lysosomal protein or real, A protein involved in endosomal vesicle trafficking leads to re locallocalization of the viral genomic RNA tracked in green to the micro tubial organization center.
Here the endosomes are tagged by lamp one and stained in red. In contrast, overexpression of an amino terminally deleted delta N that is unable to bind to P one 50 glued of the dine motor complex disperses late endosomes within the cytoplasm over expression of P 50 dyin blocks the large subunit of dine motor and releases late endosomes, but also the viral genomic RNA and HIV V one structural proteins to the cell periphery. When HIV one is expressed in helo cells and then the cells are collected and analyzed at different time points, viral RNA appears in the cell as early as three hours post transfection and infection and becomes readily observable by this fish technique by 12 hours.
Here viral RNA expression is tracked in green while the cellular protein H-N-R-N-P-A one is stained in red Once mastered, this technique can be done in 20 minutes on day one and four and a half hours on day two if it is performed properly. While attempting this procedure, it's important to remember to handle the cover slips delicately to avoid breaking them and to ensure you avoid light once the secondary antibody has been applied. Don't forget when working with HIV V one and any other higher level pathogens, it's extremely hazardous prior to fixation and precautions such as adequate training and containment should always be taken while performing this procedure.
