We have introduced a trouble shooting guide below that address common problems and feedback that we have received from beta testers and customers. The trouble shooting guide describes the sources of each problem and potential solutions.
I can’t image past 500-800 µm. Labeling appears uneven, and drops off significantly at this depth.
- Antibody concentration too high: ring of intense staining near surface, drops off significantly after that.
- Solution: Reduce antibody concentration, if signal is too weak, incubate in lower concentration for half of time, and then re-incubate in higher concentration.
- Antibody concentration too low: signal drops off into middle of tissue.
- Solution: Increase antibody concentration.
- Optical attenuation due to absorption of photons by upper layers of tissue causes “shadow” to tissues below, even with perfect staining
- Solution: Increase laser power and gain as depth increases. Caution: increased laser power increases rate of photobleaching, be sure samples contain no air bubbles. Leica SP5 and SP8 can automate laser power and gain corrections. Compare intensity loss to nuclear stain intensity, since nuclear stain diffuses very fast into tissue. Can use this signal to correct for signal loss in image processing.
Intense band of labeled tissue at surface, then significant drop-off afterwards.
- Antibody concentration too high.
- Solution: reduce concentration by increasing dilution factor. See our antibody optimization protocol.
My tissue didn’t clear!
- Plastic Incompatibility
- Visikol HISTO-2 will degrade polystyrene. For processing tissues with the Visikol HISTO approach we suggest moving away from polystyrene and towards polypropylene and glass where possible. Plastic leaching into your sample may affect the clearing ability of Visikol HISTO.
- Incomplete Dehydration/Clearing
- Most of the time a lack of tissue transparency is simply due to not completely dehydrating a sample. If you use methanol or ethanol for dehydration that has water in it and is not pure, you will not remove all the water from your tissue, resulting in tissue cloudiness. This can also be caused by not sealing the vessels containing your sample when clearing, as Visikol HISTO-2 is hygroscopic. Additionally, not using enough volume of Visikol HISTO-1 and Visikol HISTO-2 for your tissue size can cause inadequate clearing.
For a mouse brain that is not completely clear, we suggest placing the brain back into 7-10 mL of Visikol HISTO-1 for 24 hours, followed by transfer to 25 mL methanol for 2 hours. Then transfer back to 7-10 mL Visikol HISTO-1 for 24 hours, followed by 7-10 mL of fresh Visikol HISTO-2 for 24 hours.
Fluorescent protein quenched
- To visualize fluorescent protein, substitute ethanol wherever methanol appears in the protocol, and perform all steps at 4°C.
- Keep cleared samples in the dark, and protect your specimens with aluminum foil as fluorescent proteins photobleach rapidly when exposed to ambient light.
- Do not treat fluorescent protein labeled samples with H2O2 bleaching step; this step will oxidize fluorescent protein and signal will be lost.
My antibody didn’t label the tissue
- Some antibodies are not compatible with 3D immunolabeling. Validate the specificity of your antibody on small tissue sections before proceeding to larger tissues. Contact us if you have any questions about your specific antibody.
- Only purchase antibodies that have been validated for use in IHC.
- Please follow our antibody optimization protocol.
Center of tissue appears dark
- Insufficient antibody concentration
- Solution: Increase antibody concentration. A range of concentrations should be explored on a small section of tissue prior to scaling to large tissues.
- Optical attenuation leads to diminished signal at increasing depths depending on several factors, such as concentration of label bound in upper layers of tissue, level of autofluorescence, type of objective, and laser power.
Tissue has become yellow
Visikol HISTO will cause tissues to become slightly yellow during the clearing process. To reduce this effect, conduct all tissue processing steps at room temperature. This tissue yellowing will effect the orange color channel (TRITC) the most.