TITLE: Click histochemistry for whole-mount staining of brain structures
AUTHORS: Alexander Lazutkin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced.</div><div class = "text-block">• New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.</div><div class = "text-block">• Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.</div><div class = "text-block">• Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.</div></div>

[STEPS]
?. [Tissue sample preparation]
Inject animal with a synthetic thymidine analogue 5-ethynyl-2’-deoxyuridine at a dose of 40-123 mg/kg. The time to euthanasia may vary depending on the specific research question.
?. [Tissue sample preparation]
Anesthetize animal with chloral hydrate (10 mg/kg) immediately before euthanasia.
?. [Tissue sample preparation]
Perfuse animal intracardially with 30 ml of PBS and 30 ml of cold 4% paraformaldehyde, pH 7.4.
Paraformaldehyde requires careful handling since it is highly toxic and potentially carcinogenic. Work only in a fume hood .
?. [Tissue sample preparation]
Dissect the brain in 1 h after perfusion.
?. [Tissue sample preparation]
Postfix the brain samples by overnight (ON) immersion in 4% PFA at .
4 °C
?. [Tissue sample preparation]
Rinse the brain twice with PBS for 2 h each time.
?. [Tissue sample preparation]
Remove the cerebral cortex from the fixed hemispheres using a microsurgical spatula, then isolate the hippocampi and place it in 2 ml microtube with cold PBS.
?. [Pretreatment and storage]
Incubate the specimen in Dent solution (100% methanol/DMSO, in the ratio of 4:1) for 2 h at .
4 °C
Methanol requires careful handling since it is highly toxic and highly inflammable liquid.The methanol/DMSO solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.
?. [Pretreatment and storage]
To destroy endogenous pigments, bleach the specimen in Dent bleach solution (100% methanol/DMSO/30% H2O2, in the ratio of 4:1:1) in bright light for 2 h until the samples are completely white.
?. [Pretreatment and storage]
After bleaching, rinse the specimen three times in 100% methanol for 1 h each time.
?. [Pretreatment and storage]
At this stage, specimens can be stored in 100% methanol at . It is important to warm up the preparations gradually after storage to preserve the morphology. For this, transfer the sample first to , then to , and only after that brought it to .
-70 °C
-20 °C
4 °C
4 Room temperature
?. [Pretreatment and storage]
Rehydrate samples stepwise in 75%, 50%, 25% and PBS for 1 h in each solution.
?. [Pretreatment and storage]
Wash rehydrated sample twice in PBS for 1 h each time.
?. [Pretreatment and storage]
Permeabilize the preparation in 2% saponin and 5% DMSO solution in PBS for 1 h at .
37 °C
?. [Pretreatment and storage]
After permeabilization, wash the preparation in Tris-HCl buffer (pH 8.0) with 0.2% Triton X-100 for 1 h.
?. [Click reaction]
Incubate the specimen in click reaction solution (Tris-HCl buffer containing 5% DMSO, 0.2% Triton X-100, 0.2% saponin, 100 mM sodium ascorbate, 1 mM CuSO4 and 10 mM fluorescent azide) for 2 h in the dark.
Click reaction solution must be prepared immediately before staining. 1 M sodium ascorbate stock solution must be prepared from the powder immediately before the click reaction.
?. [Click reaction]
Stop the click reaction by three 1 h incubations in Tris-HCl buffer with 5% DMSO, 0.2% Triton X-100 and 0.1 M EDTA.
?. [Click reaction]
Wash stained preparation twice for 1 h with PBS containing 5% DMSO and 0.2% Triton X-100 and twice with PBS for 1 h each time. Final rinsing with PBS was done overnight at .
4 °C
?. [Clearing]
Dehydrate the preparation in 25%, 50%, and 75% methanol and three times in 100% methanol for 1 h for each incubation.
?. [Clearing]
Clear the preparation for ON in dibenzyl ether (DBE) in the dark at . Samples can be stored in clearing solution.
4 °C
Dibenzyl ether requires careful handling since it is strong non-polar solvent. Use only compatible plastic (polyethylene etc.) and glass labware.