The overall goal of this procedure is to prepare Arabidopsis Vues for whole mount hybridization and immuno staining. This is accomplished by first fixing fresh flower buds in the fixative solution. The second step is to carefully dissect and embed vues in miniature acrylamide pads directly on microscopy slides.
Next tissue processing enables tissue clarification and permeation. The final step is to incubate the treated samples with an antibody solution for immuno staining or a labeled probe for fluorescent in C two hybridization. Ultimately, high resolution confocal imaging followed by three dimensional reconstruction, allows for quantitative analysis in whole mount at the single cell level.
This method was initially employed to provide insight into CT organization in the view, but it could also be applied to analyze other cell compartments in other tissues of the plunge and in other model organisms Into micro fuge tubes filled with freshly made BBO buffer chilled on ice at 20 to 30 carpals per tube set the tubes to rock gently at room temperature for half an hour. Next, spin down the tubes for one minute at 400 Gs.Then carefully aspirate the supernatants, discard them, and add a milliliter of PBT to the carpals. Place the tubes on ice for each tube of carpals to mount.
Have prepared on ice. Five tubes containing 200 microliters. A freshly made 5%acrylamide mix.
Five clean super frost slides labeled with a pencil, a thought Eloqua of 20%a PS and A thought Eloqua of 20%NAPS from one tube of carpals. Take four or five with a cut end tip and place them each onto one slide. Remove the excess liquid by pipetting carefully using fine needles.
Make longitudinal cuts in the carpals and take away the carpal walls to release the rows of vues. This step will take practice. Prevent the vues from drying by adding up to 10 microliters of PBS, then to a micro fuge tube containing the acrylamide mix.
Quickly add 12 microliters of NAPS and 12 microliters of a PS.Mix the solution well with pipetting and quickly add 30 microliters of this activated acrylamide. Mix onto a slide with dissected vues. Gently place a small cover slip on the preparation and allow the solution to polymerize at room temperature for about an hour.
Prepare each slide in this manner later. Use a razor blade to chip off the cover slips. The samples can be stored overnight at four degrees Celsius in a coplan jar with PBS or proceed directly with processing.
Generally processing should be carried out in coplan jars with 80 milliliters of solution. And under the fume hood. Begin by clarifying the tissues through incubation in a solution series of methanol ethanol, a one-to-one ethanol xylene solution, and again in ethanol and then methanol.
Each bath is five or 30 minutes. See the text protocol. Next, use a one-to-one methanol PBT solution with 2.5%formaldehyde for 15 minutes.
Then rinse the tissue in PBT twice for 10 minutes per bath. After this, the slides can be stored overnight at four degrees Celsius if necessary. Next, take up to six slides from the jar and drain off the excess liquid.
Place them onto a tissue paper and quickly add 100 microliters of a chilled cell wall digestion enzyme. Mix onto the pads. Then cover the slides with large cover slips.
Incubate the slides for two hours at 37 degrees Celsius in a moist chamber. It is important to optimize the CO digestion temp for every new enzymatic solution because this step is critical for a homogeneous staining Later. Wash the slides twice with PBT for five minutes per wash.
Then drain the slides now to each slide. Add 100 microliters of RNAs A in PBS completed with 1%tween and incubate the slides for an hour at 37 degrees Celsius in a moist chamber after the incubation, wash the slides twice with PBT as before, and then fix the tissue again by incubating the slides in a bath of freshly made PBTF for 20 minutes. Rinse the PBTF off with one wash in PBT for 10 minutes.
Then proceed to tissue permeation by incubating the slides in PBS with 2%tween for two hours at four degrees Celsius. Use two washes in PBT each of five minutes to remove the permeable solution. Then proceed with immuno staining.
Begin with incubating the slides in the primary antibody of interest. Apply 100 microliter aliquots of the antibody diluted in PBS and with 0.2%between to each slide. Transfer the slides to a moist chamber and incubate them at four degrees Celsius for 12 to 24 hours.
Wash the slides in a bath of PBT for two to four hours with gentle rocking at room temperature. Next, add 100 microliters of secondary antibody diluted one to 200 in PBS with 0.2%between 20 and incubate the slides for 24 hours at four degree Celsius. A one hour bath in PBT with gentle rocking is sufficient to wash the slides of unbound secondary antibody.
Then counterstain the DNA with a solution of 10 micrograms per milliliter of propidium iodide in PBS. Allow the staining to carry on for 15 minutes and then wash the slides with PBT using gentle rocking for 15 minutes at room temperature. Now mount the slides with an anti fade medium supplemented with 10 micrograms per milliliter of propidium iodide.
After letting set for an hour image the slides on a confocal microscope using a 63 x glycerol immersion lens. So during image acquisition, it is critical to maintain the scanning settings identical between samples to enable comparative quantification and to use a linear detection mode. Later reconstruct the serial images into three dimensional structures, segment each nucleus of interest and use 3D image processing software to quantify the signals.
Using this robust large scale preparation protocol, whole Mount Vues will retain their three-dimensional structure. Subcellular structures become visible with high detail as seen with chromatin. When staining DNA hetero chromatin appears as a bright and well-defined foci without the need to apply deconvolution.
This protocol permits parallel immuno staining with several antibodies in one round. For example, the U chromatin associated permissive marker, H three trimm methylated lysine four, and the hetero chromatin associated marker H three monomethyl lysine 27 were both detected in one experiment on distinct replicate slides to analyze chromatin dynamics. Another compatible technique is fluorescent C two hybridization or fish fish to 45 s.Ribosomal.
DNA repeats was used to define nuclear organization regions. The probe was directly labeled with Alexa 4 88, and the DNA was counters stained For ification analysis, it's very important to test different and body dilutions and incubation times to identify the conditions that giving robust and hogene signal with the lowest possible. And body contrition.
As this method achieves excellent preservation of the tissue and optimal permeation for single cell analysis of chromatin organization, it paves the way for scientists in the field of plant cell biology or plant epi epigenetics to investigate nuclear or subcellular organization at a single cell level and in the whole man context.
