A mechanic discovered a mouse in a barrel of degreaser

There are many different protocols for clearing tissues as preparation before staining and imaging.

I use a modified CUBIC protocol as well as other protocols for clearing insect brains that I later stain with fluorescent dye and scan with a confocal microscope before making a 3D reconstruction of the brains in a special software. It’s super cool, especially as the techniques can be used for whole animals.

About CUBIC: https://en.m.wikipedia.org/wiki/CUBIC It’s a bit technical, but explains the process a bit.

The opacity of brain tissue is given mainly by light scattering on interfaces between environments with various refractive indexes, mainly between lipids and other tissue compounds.[5] Therefore, partial delipidating and refractive index matching of tissue and surrounding medium is straightforward way to make tissue less opaque and therefore transparent – cleared.[1]

Development of CUBIC pipeline was inspired by previously published clearing protocol named Scale (mixture of glycerol, urea and detergent), because of its simplicity and optimal compatibility with fluorescent proteins.[6] Authors of CUBIC screened 40 chemicals corresponding to those used in Scale with aim to conserve compatibility with fluorescent reporters but achieve better and faster clearing of the tissue. They found that basic amino alcohols are ideally suited for this purpose, probably because amino groups effectively solvate phospholipids and basicity helps to preserve fluorescence signal.[1] Amino alcohols have also beneficial effect when used for clearing of other tissues, which are mostly highly vascularized, and their opacity is given by absorption of light by hemoglobin on top of light scattering. Amino alcohols reduce pigmentation of those tissues very effectively by eluting the hem from hemoglobin.[7]

The original protocol[7] is two-step incubation of fixed tissue in two different aqueous based clearing solutions, altogether taking one to two weeks. First solution, referred as ScaleCUBIC-1, CUBIC-1 or just reagent-1, is composed of N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (commercially under name Quadrol), urea and Triton X-100 in water. Second solution, referred as ScaleCUBIC-2, CUBIC-2 or just reagent-2, is composed of urea and sucrose in water.[8] This original protocol is slightly modified in different applications, namely in concentrations, incubation times or some components of solutions.[7][9][10] The CUBIC protocol can be also combined with perfusion and provide whole organ and whole body clearing of rodents.[7] Besides its use as standalone protocol for clearing, CUBIC-based composition of reagents can be used as refractive index matching solution for CLARITY.[4] This than improve clearing abilities of CLARITY on tissues which stay opaque because of their pigmentation by hemoglobin.

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