The overall goal of this procedure is to illustrate how to prepare live tissue slices for imaging pheromone responses in MRO nasal organ neurons. This is accomplished by first preparing live MRO nasal organ or VNO tissue slices from mice expressing a genetic calcium indicator GCaMP two. The next step is to set up the perfusion system on the microscope stage for maintaining the slice, applying stimulus and imaging.
The response. A time-lapse imaging experiment is then performed with various pheromone stimuli, followed by data analysis of the imaging results. Ultimately, response patterns of VNO neurons to different pheromone stimuli are obtained.
The main advantage of this technique or existing methods like calcium D loading or imaging in dissociated cells is that we can preserve the intact morphology of the cells, better maintain the health of the slices, and obtain a stronger signal. This method can help us to answer some of the key questions in the studies of warm nasal system, such as identifying the ligands and their receptors and profiling the individual worm worm nasal neuron responses. This method can also be applied to other systems such as olfactory, serum, or brain psoriasis.
Generally, individuals new to this method may struggle because of the difficulty in obtaining good psoriasis and setting up the profusion system. Before beginning this procedure, prepare the required solutions, including the mouse, artificial cerebral spinal fluid ringer solution, 4%low melting aros and pheromone stimuli according to the accompanied manuscript. Then put two tubes of low melting aros on a heat block.
Set the temperature to higher than 60 degrees Celsius to melt the aros. Once the gel liquefies transfer the tubes to a 37 degree Celsius heat block. To prepare the VNO slices.
After decapitating a euthanized GCaMP two mouse, cut the mandible bones with a pair of scissors to remove the lower jaw. Next, peel off the ridged upper palate tissue to expose the nasal cavity. Then separate the jawbones by inserting a surgical blade between the two front incisors.
Carefully remove the jawbone to expose VNO. At this point, lift the whole VNO up from the nasal cavity by holding on the tailbone. Transfer the VNO to the cold oxygenated mouse, A CSF solution.
Immediately under the dissection scope, separate the two vno by gently sliding the tips of the forceps along the wall of septal bone. Peel away the vulner bone that encases the VNO tissue. Then gently lift the entire VI NO tissue from the bone cavity.
Extreme care should be taken in this step to avoid damaging the neuro epithelium. Next, remove the small bone fragments left on the tissue surface completely. Before embedding fragments left on.
The tissue may be caught by the cutting blade that would pull the tissue out of the aros block. After that, use the forceps to hold the posterior end of the VNO tissue and gently submerge it into the melted aros. Quickly cool the tube on ice.
To solidify the aros, the embedding and cooling process should take less than two minutes. Then trim the aros block containing the VNO and glue it to the tissue holder. Subsequently, insert the tissue holder into the metal cylinder and fill the remaining chamber with additional low melting aros.
Once the low melting aros solidifies on ice, proceed to sectioning immediately supply cold oxygenated mouse A CSF to the sectioning chamber of the tissue slicer, and cut the slices at 180 to 200 micron thickness each. Then collect and transfer the section slices to the incubation chamber. The slices are viable for six to eight hours in the oxygenated mouse A CSF at room temperature.
In this procedure, first place a VNO slice in the middle of the perfusion chamber and hold the slice down with a slice anchor. The threads of the anchor should only press against the low melting aros part of the slice, but not the VNO tissue. Then deliver the oxygenated mouse A CSF to the perfusion chamber through the inlet port at about 100 microliters per second.
The liquid is drained through a suction needle via the outlet port to provide a continuous flow of fresh mouse A CSF. Next, fill a 30 milliliter syringe with ringer solution and clamp it to the syringe pump. Set the pump speed to 300 to 600 microliters per minute.
To provide a continuous flow of ringer solution over the slice, connect the outlet of the ringers to A-H-P-L-C injection loop. The injection loop control has two flow routes. At the load position, load the sample of mouse urine into the sample loop with a Hamilton precision syringe.
Excess sample exits the sample loop through the waste outlet. Ringer's solution injected by the syringe pump bypasses the sample loop and goes directly to the outlet, which is connected to the perfusion tip at the injection position. The pump solution flows through the sample loop and pushes the pheromone stimuli into the outlet.
Prior to the imaging experiment. Remove the air bubbles to ensure smooth flow of the perfusion fluid. Then adjust the perfusion tip under a five x or 10 x lens so that the tip is about one millimeter away from the vien O slice.
To measure the sample delay time and the duration with fluorescent dye load 0.1%rho domine six G dye into the sample loop. Then switch the valve to the injection position. Five seconds after the start of image acquisition, detect the fluorescent signal between 10 to 30 seconds.
The smooth curve of the fluorescent signal also indicates that there is little turbulence generated under the dipping lens and that adequate perfusion of oxygenated mouse A CSF reaches the slice In our laboratory. The Zeiss Axio scope FS two microscope with a 10 x or 20 x water dipping lens is used for time-lapse imaging for detecting GAMP two signals. The standard GFP band pass filter of 450 to 490 nanometers is used.
The epi fluorescent images are acquired by a CCD camera with one by one or two by two spinning depending on the expression levels of GCaMP two. First, set the acquisition speed to one frame per second. Adjust the intensity of the light to minimize bleaching of the GCaMP two signals and photo damage to the cells.
Then switch the valve to the injection position at a specific time point so that consistent time delays in all trials are obtained within a set of experiment. Now, perform a test run using ringer's solution as the stimulus readjust the perfusion setup if movement artifact is introduced during the sample injection. After that, perform a positive control run with the mouse urine diluted at one to 100 in solution to prevent cross-contamination among different samples.
Wash the Hamilton syringe with ringer solution at least three times after loading one stimulus. Similarly, wash the sample loop with ringer's solution at least three times between different stimuli before applying. The next stimulus wait for four to 10 minutes for the MRO nasal sensory neurons to recover to perform image registration of all the images acquired from one slice.
A custom written VBA script in Avision is used to automate this process. In this case, all the image frames within the same experiment are registered against a common chosen reference frame with elastic to perform image subtraction to identify the responding cells. The custom written macros in image J version 1.42 are used to automate this process.
A minimal projection image is generated for each stack and the responding cells emerge after the minimal projection is subtracted from the raw stacks. Next, identify the region of interest from the subtracted stacks and obtain the ROI coordinates using multi mesure plugin from Image J.Then process all the stacks for one experiment and save all the ROI coordinates in a ROI master list. After that, use the ROI master list to measure the cell responses from the raw image stacks with custom written macro and multi mesure plugin from image J.Then plot the response curves and heat map in matlab.
Shown here is an example of the VNO imaging experiment using the urine samples collected from four individual mice. About 80 cells show response to at least one of the urine stimuli and their response delta F divided by F values are plotted in the heat map. Here are the response traces of cells one, two, and three, which show the time course activation by urine stimuli cell one displays response to both female urine samples, but not the male samples.
Whereas cell two shows the opposite patterns of activation and responds to both male samples. Only cell three is activated by both individual male and female samples. Once mast, the slice can be prepared in 30 minutes and the piano neurons can be kept alive for six to eight hours if it is performed properly.
While attempting this method, it is important to remember to use tissue cultural grade reagents and to and avoid tissue fixation reagents in contacting with the tissue preps. After watching this video, you should have a good understanding of how to prepare live tissue psoriasis for imaging thermal responses in Vienna neurons using mice expression G Camp.
