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Background Information
Background Information
Photobleaching is an optional step which helps remove pigment and other fluorescence which can interfere with imaging. This step is necessary in samples with significant pigment, such as eyes or spleen, and is extremely useful in situations where the animals cannot be perfused or where there is significant lipofuscin. It can also be used to remove signal after imaging to prepare the sample for subsequent rounds of labeling and imaging.
In the example to the right we started with PFA fixed mouse brains which were not perfused and contain a lot of blood, and mouse hearts which are typically quite pigmented. All samples were bleached overnight in the same solution described below and either with no light, the advanced setup described below, or the simple setup described below. It is possible to fully bleach samples without light, but it just takes longer and may require refreshing the bleaching solution. Please experiment with the different setups as you see fit!
Reagents / Equipment Required:
Reagents / Equipment Required:
Sodium percarbonate. (Sigma 371432, or Chem Impex 35873)
Sodium azide (Sigma S2002)
10X PBS
Photobleaching setup, 2 options are described below.
Protocol
Protocol
[Note] This type of photobleaching is indiscriminate - it will reduce all fluorescence in the sample including endogenous fluorescent reporters and dyes!
Prepare a solution of 0.5% Sodium azide and 0.5X PBS in distilled or deionized water.
Add solid sodium percarbonate to the solution to a final concentration of 0.5% m/v and vortex until fully dissolved. It is important to prepare this fresh! Using sodium percarbonate ensures the bleaching solution is always fresh and at known concentration. The sodium azide in the solution helps prevent excessive oxygen bubble formation which can cause problems inside the samples.
Incubate the sample in this solution in the LED photobleaching setup at 4°C until it is white and decolorized. We typically bleach samples overnight, but the duration is dependent on the sample type and light intensity. Bleaching without the light is possible, but it is slower and less effective.
After bleaching, wash the sample with PBS+0.02% sodium azide at 4°C for several hours before moving to the next step in your protocol which is most likely SHIELD fixation.
Simple Photobleaching Setup
Simple Photobleaching Setup
This is a simple setup that is cheap to build and relatively effective.
Equipment Required:
Equipment Required:
A beaker large enough to fit your sample tube of choice.
An adhesive-backed white LED strip. In the example we used this one from Amazon.
Aluminum foil.
The Build
The Build
Open the LED strip and apply in a spiral pattern to the beaker. If it is too difficult to apply to the inside of the beaker, you can attach them to the outside of the beaker facing outward. Once it is wrapped, cut the strip to length.
Wrap the outside of the beaker in aluminum foil.
When you are ready to use the photobleacher, put the sample tube inside the beaker and cover the front with aluminum foil. It's best to run the photobleacher in the fridge or in a cold room as the sample will warm up!
Advanced Photobleaching Setup
Advanced Photobleaching Setup
Equipment Required:
Equipment Required:
3D Print a frame. Any rigid material will do.
12V LED power supply. A waterproof version is ideal in the case of spills. This power supply has excess power, is IP67 rated and has 2 outputs which makes setup easier.
140 mm cpu fan. Any fan this size will do.
If your fridge has accessible power inside and you don't need to run a cable through the door, a wireless outlet like this one is useful to turn the lights on and off.
The Build
The Build
Attach the male barrel plug connectors to the power supply outputs and the female barrel plug connector to the light. Make sure to strip the wires properly before attaching to the screw terminals, and match the red wires to the (+) terminals and black wires to (-) as shown.
Print the frame and use the screws provided with the fan to mount it to the frame as shown. It's best to orient the fan to push air into the middle of the frame.
Slide the light into the frame from the open side opposite the fan with the lights facing up.
Plug the light and fan into the power supply.
Move the whole setup to the fridge or cold room. You can use a wireless outlet like the one linked above as a switch to turn the light on and off.
[Note] This light is very bright! Don't look directly into it!
[Note] Don't put this setup inside a mini fridge. It may not have the cooling capacity to keep up with the heat generated.The frame is designed to hold standard well plates. You can bleach directly in the well plate, but make sure to cover the top with well plate film to prevent evaporation of liquid. We typically bleach in 5 mL tubes laying inside a monowell plate. The screwcap prevents leaks and evaporation. Before turning on the light, place a plastic mirror on top of the well plate to reflect light back down at the samples.
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