Visikol HISTO Beta Test Feedback - Tissue Yellowing and Background Fluorescence

Tissue Yellowing

Some researchers have expressed that tissues are generally yellower than they expected following clearing with the Visikol HISTO approach. This expectation is due in part to what researchers are familiar to seeing with the CLARITY approach wherein tissues lack any pigmentation. Because Visikol HISTO clears tissues through the process of refractive index matching and not removing cellular components (lipids) like the CLARITY technique, some tissue pigmentation will remain that appears yellow. This pigmentation should not interfere with imaging , but can be reduced through tissue processing.

Pigmentation can be reduced through using tissues that have been perfused and/or bleaching tissues with 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4°C overnight.

Rat brain cleared with Visikol HISTO

Background Fluorescence

One of the other topics that has come up from some researchers that is related to tissue pigmentation is background fluorescence. Researchers have seen higher than expected background fluorescence in tissues that have been cleared with Visikol HISTO. This background fluorescence is due to three primary causes: 1) tissue fixation, 2) heme pigment and 3) endogenous pigments (lipofuscin, collagen). The guidance below can assist with reducing background fluorescence and choosing the best fluorophores for imaging. 

1) Tissue Fixation

Cross-linking of proteins (tyrosine and tryptophan) with formaldehyde generates increased autofluorescence due to formaldehyde adducts. Glutaraldehyde causes this issue even more. The best way to avoid autofluorescence due to fixation is to be sure to fix tissues for the minimum amount of time required for the size and type of tissue. Generally tissues should be fixed in freshly prepared 4% PFA (paraformaldehyde) / 10% NBF (neutral buffered formalin) overnight at 4°C and then at room temperature for 1-2 hours. Larger tissues may require longer incubation times, but in general tissues immersion fixed should be less than 6 mm thick. After fixation, tissues should be transferred to PBS containing 0.01% sodium azide or similar preservative, and stored at 4°C. 

While generally intrinsic to most tissues, this can be avoided by using non-crosslinking fixatives. Non-crosslinking fixatives will also increase the ability of antibodies and stains to penetrate the tissues. The use of non-crosslinking fixatives may lead to loss of some epitopes if the protein of interest is not bound to the structure of the tissues. Email us at info@visikol.com to discuss your specific tissue and the most optimal fixation method. 

2) Heme Pigment 

The best way to deal with heme is simply to perfuse tissues with PBS prior to fixation at the point of sacrifice. This procedure will remove the majority of blood cells from tissues and eliminate this problem at the source. If it is not possible to perfuse the tissue (i.e. archived specimens), there are techniques that can be applied to bleach the tissue and remove complications from the heme. The procedure involves incubating tissues in 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4°C overnight prior to staining and clearing steps.

3) Endogenous pigment

Lipofuscin is a lipophilic pigment that accumulates through normal aging processes in animal tissue and is significantly autofluorescent. Lipofuscin often appears as small yellow granules in fluorescent imaging. 

Collagen is a structural protein which occurs in large amounts in many tissues (e.g. liver, muscle) and is strongly autofluorescent in the blue region (350-450 nm), and lesser so in the green region (475-550 nm). 

There are several other biomolecules that are innate to animal tissue which promote autofluorescence. These include nicotinamides (NADP), retinols and carotenoids, bile acids and downstream products (bilirubin).

To address autofluorescence derived from these sources, bleaching tissues with peroxide can be effective. The procedure involves incubating tissues in 5% H2O2 in Methanol/DMSO (1 part 30% H2O2, 4 parts methanol, 1 part 100% DMSO) at 4°C overnight prior to staining and clearing steps

Reach out to us about your specific tissue and auto-fluorescence problem and we will work with you to address it (info@visikol.com).

Imaging Guidance - Choosing Fluorophores

Many of the problems arising from strongly autofluorescent tissues can be circumvented by a wise strategy for choosing fluorophores. A wide range of chemical fluorophores have demonstrated compatibility with Visikol HISTO, such as the AlexaFluor dyes, Cy dye series, Atto dyes, FITC, Texas Red, DAPI, Hoechst 33342, Propidium iodide, DRAQ5, SYTOX dyes and many others. Apart from fluorescent proteins. Visikol HISTO has not been shown to interact, quench, or diminish fluorescence in any commonly used fluorophores. There is a simple strategy for maximizing results of fluorescent labeling.

Highest priority targets should be paired with red fluorophores (e.g. AlexaFluor 647), next highest targets paired with yellow fluorophore (e.g. AlexaFluor 596), third priority targets with green fluorophore (e.g. AlexaFluor488), and nuclear staining / broadly expressing epitopes labeled with blue fluorophores. The logic behind this strategy is simple, autofluorescence is highest in blue, and decreases towards red, and therefore fluorophore priority goes from red to blue.

Fluorophore Priorities by Color

Spectrum.png

Lower priority targets (e.g. nuclear staining)

 

Medium priority targets (Structural markers, higher expressed epitopes)

 

High priority targets (low expressed epitopes, important biomarkers, etc.)