Tissue Clearing Kit

Photos were taken before and after mouse brains were cleared following five different tissue clearing protocols:

• Hello Bio: Cleared using HB8771 following the accompanying tissue clearing protocol 
• iDISCO: Cleared following protocol detailed in Renier et al., 2014
• Ub1: Cleared following protocol detailed in Hough et al., 2021
• UbasM: Cleared following protocol detailed in Chen et al., 2017 
• AKS: Cleared following protocol detailed in Shan et al., 2022

Maximum intensity projection of tyrosine hydroxylase staining (HB6605) in a cleared mouse brain segment. Method: A mouse brain was dissected and fixed in 4% PFA for 24 hours before washing and incubation in 30% sucrose for 3 days before being dissected into smaller segments for clearing. The brain segment was incubated in solution A for 3 days at 37°C before washing and incubation in solution B for 3 days. After washing, the tissue was blocked for 3 days then incubated in primary antibody (rabbit polyclonal anti-tyrosine hydroxylase (HB6605)) at a 1:500 dilution for 3 days. Following washing, the tissue was incubated with a goat anti-rabbit DyLight 650 conjugated secondary antibody (1:300 dilution) for a further 3 days. Following more washing the tissue was incubated in mounting solution for 3 days until clear then imaged using a confocal laser scanning microscope to create a z-stack. This was then flattened in ImageJ using the maximum z-projection function. For more information please see our tissue clearing protocol.

Maximum intensity projection of tyrosine hydroxylase staining (HB6605) in a cleared mouse brain segment. Method: A mouse brain was dissected and fixed in 4% PFA for 24 hours before washing and incubation in 30% sucrose for 3 days before being dissected into smaller segments for clearing. The brain segment was incubated in solution A for 3 days at 37°C before washing and incubation in solution B for 3 days. After washing, the tissue was blocked for 3 days then incubated in primary antibody (rabbit polyclonal anti-tyrosine hydroxylase (HB6605)) at a 1:500 dilution for 3 days. Following washing, the tissue was incubated with a goat anti-rabbit DyLight 650 conjugated secondary antibody (1:300 dilution) for a further 3 days. Following more washing the tissue was incubated in mounting solution for 3 days until clear then imaged using a confocal laser scanning microscope to create a z-stack. This was then flattened in ImageJ using the maximum z-projection function. For more information please see our tissue clearing protocol.

Section of cleared rat hippocampus stained for GFAP (HB8267) and Neurofilament L (HB7266) alongside DAPI counterstaining. Method: Rat hippocampi were dissected from fresh brains and fixed in 4% PFA for 12 hours before washing and incubation in 30% sucrose for 3 days. The hippocampi were incubated in 5ml of solution A for 2 days at 37°C before washing and incubation in 5ml of solution B for 2 days. After washing, the hippocampi were blocked for 3 days then incubated in primary antibodies (mouse anti-GFAP (HB8267) at a 1:200 dilution (5µg/ml) and rabbit anti-neurofilament L (HB7266) at a 1:3,00 dilution (3.3µg/ml) for 1 week. After more washing, the hippocampi were then incubated in secondary antibodies for 6 days (goat anti-mouse Dy488 and goat anti-rabbit Dy594, both at 1:200). Finally, following washing the hippocampi were incubated in DAPI for 3 hours and then washed and incubated in storage solution for 2 days. Images were captured using a Zeiss Z.1 lightsheet microscope. For more information please see our tissue clearing protocol. for 1 week. After more washing, the hippocampi were then incubated in secondary antibodies for 6 days (goat anti-mouse Dy488 and goat anti-rabbit Dy594, both at 1:200). Finally, following washing the hippocampi were incubated in DAPI for 3 hours and then washed and incubated in storage solution for 2 days. Images were captured using a Zeiss Z.1 lightsheet microscope.

Maximum intensity projection of tyrosine hydroxylase staining (HB6605) in a cleared mouse brain segment showing dopaminergic axons in the caudate putamen. Method: A mouse brain was dissected and fixed in 4% PFA for 24 hours before washing and incubation in 30% sucrose for 3 days before being dissected into smaller segments for clearing. The brain segment was incubated in solution A for 3 days at 37°C before washing and incubation in solution B for 3 days. After washing, the tissue was blocked for 3 days then incubated in primary antibody (rabbit polyclonal anti-tyrosine hydroxylase (HB6605)) at a 1:500 dilution for 3 days. Following washing, the tissue was incubated with a goat anti-rabbit DyLight 650 conjugated secondary antibody (1:300 dilution) for a further 3 days. Following more washing the tissue was incubated in mounting solution for 3 days until clear then imaged using a confocal laser scanning microscope to create a z-stack. This was then flattened in ImageJ using the maximum z-projection function. For more information please see our tissue clearing protocol.

Maximum intensity projection of tyrosine hydroxylase staining (HB6605) in a cleared mouse forebrain showing the dopaminergic neurons of the striatum. Method: A mouse brain was dissected and fixed in 4% PFA for 24 hours before washing and incubation in 30% sucrose for 3 days before being dissected into smaller segments for clearing. The brain segment was incubated in solution A for 3 days at 37°C before washing and incubation in solution B for 3 days. After washing, the tissue was blocked for 3 days then incubated in primary antibody (rabbit polyclonal anti-tyrosine hydroxylase (HB6605)) at a 1:500 dilution for 3 days. Following washing, the tissue was incubated with a goat anti-rabbit DyLight 650 conjugated secondary antibody (1:300 dilution) for a further 3 days. Following more washing the tissue was incubated in mounting solution for 3 days until clear then imaged using a confocal laser scanning microscope to create a z-stack. This was then flattened in ImageJ using the maximum z-projection function. For more information please see our tissue clearing protocol.