Basic NeuroscienceA multispectral LED array for the reduction of background autofluorescence in brain tissue

- We established a highly effective method to reduce artifactual autofluorescence (AAFs) in fixed mammalian brain tissues.
- An array of light emitting diodes (LEDs) was used to photo-irradiate the tissue and quench the autofluorescence.
- The photo-irradiation process can reduce AAFs to near tissue background levels.
- Photo-irradiation can be performed on brain specimens prepared as either slide-mounted or free-floating in-well plate sections.
- The staining quality of neural and glial markers remained robust following the LED photo-irradiation treatment.

The presence of fixative-induced and cellular-derived artifactual autofluorescences (AAFs) presents a challenge in histological analysis involving immunofluorescence. We have established a simple and highly effective method for the reduction of AAFs that are ubiquitous in fixed mammalian brain and other tissues. A compact AAF-quenching photo-irradiation device was constructed using a commercially available light emitting diode (LED) array, cooling unit, and supporting components. The LED panel is comprised of an array of multispectral high intensity LEDs which serve as the illumination source for the photo-irradiation process. Rabbit and cat brain specimens of 5 μm- and 40 μm-thicknesses, respectively, were photo-irradiated for various durations. The AAFs were reduced to near tissue background levels after 24 h of treatment for both deparaffinized and paraffinized rabbit brain specimens, and for the free-floating cat brain specimens. Subsequent immunofluorescence staining using primary antibodies against GFAP, NeuN, Iba-1, and MAP-2, and the corresponding Qdot® and Alexafluor® fluoroconjugates confirmed that the LED photo-irradiation treatment did not compromise the efficiency of the immunofluorescence labeling. The use of the device is not labor intensive, and only requires minimal tissue processing and experimental set-up time, with very low maintenance and operating costs. Finally, multiple specimens, in both slide and well-plate format, can be simultaneously photo-irradiated, thus, allowing for scalable batch processing.