Incubated with the primary antibody at 4°C overnight.

Incubated with the secondary antibody at room temperature for 1 hour.

Passive CLARITY technique (PACT)-sorbitol refractive index matching solution (sRIMS) clearing and 3D imaging of virgin and lactating mouse mammary tissue.aPACT-sRIMS tissue clearing and immunostaining protocol and timeline. Three-dimensional confocal imaging of PACT-sRIMS-cleared virgin (b) and lactating (c) mammary glands immunostained with basal cell markers (K5 and smooth muscle actin (stained withab5694)) and luminal cell markers (K8 and E-cadherin (E-CAD)).Main imageshows the maximum intensity projection of the entire image sequence, with thin optical slices (1μm) and their depth (zvalue) relative to the first image in the image sequence.

From a paper comparing imaging of intact virgin and lactating mammary glands using 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique.

This picture shows formalin-fixed, paraffin embedded mouse intestine and mesentery, the optimal dilution is 1:1600 to 1:3200, incubation overnight at 4^oC,counterstained withHematoxylin.

This image was kindly supplied as part of the review by JQ Zhang.

Pancreatic vessel imaging in the intact adult mouse pancreas.In adult mouse tissues (12weeks old), imaging was performed after CLARITY. Three-dimensional projection clarified mouse pancreas with capillary immunostained for α-smooth muscle actin (green). Scale bar, 200 μm.

From astudy, that aimed to improve the original CLARITY procedure and developed specific CLARITY protocols for various intact organs.

Representative histology of aortic valve leaflets from aged mice demonstrates changes in pRb cKO AoV

A) Masson’s trichrome showing reduced collagen staining (blue) in leaflet from pRb cKO mouse with aortic regurgitation (AR). B) Movat pentachrome showing more diffuse collage staining (yellow) in fibrosa, but normal proteoglycan staining (blue) in the spongiosa layer of the leaflet from pRb cKO with AR. C) Immunohistochemistry for α-SMA, demonstrating presence of activated myofibroblasts throughout leaflets of pRb cKO mouse with and without AR. Scale bar is 50μm.

Alpha smooth muscle Actin is detected witn ab5694 at 1/1000 dilution.

(From Figure 2 of Freytsis et al)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling alpha smooth muscle Actin with ab5694 at a dilution of 1/1000. Heat mediated antigen retrieval was performed for 35 minutes followed by cooling for 20 minutes. Sections were incubated with the primary antibody for 1 hour followed by incubation with a biotinylated secondary antibody for 30 minutes then HRP-Streptavidin for 30 minutes. Developed using DAB chromogen substrate (5-10 minutes). Counter stained with hematoxylin.

Magnification: left - 10X,right -40X.