Figure 3.

Improved permeabilization-free immunostaining with the preservation of ECS

(A) Detergent-free immunolabeling of NeuN in mouse cerebral cortex using antibodies.

(B and C) YFP-H mouse brain tissues were stained with fluorescent anti-GFP antibodies to compare antibody penetration. Images were acquired at cross-sections to demonstrate the antibody penetration depth after 5 days of permeabilization-free immunostaining at 4°C. The results revealed that anti-GFP antibodies penetrated approximately 60 μm in ECS-preserved brain tissues (B) and less than 10 μm in ECS-absent tissues (C).

(D) PV+ interneurons in the cortex were labeled with fluorescent scFv. The light blue arrows show the PV+ interneuronal synaptic boutons (red) surrounding large pyramidal cell somata (unlabeled).

(E) Labeling of PV+ neurons with antibodies (red) and perineuronal nets (PNNs) with biotinylated wisteria floribunda agglutinin (bWFA; yellow) in the cortex. X-Z and Y-Z reslices show labeling was throughout the imaged depth of the ECS-preserved brain tissue.

(F and G) Immunolabeling of PSD-95 (magenta) in the cerebellar cortex is similar with permeabilization (F) and without permeabilization (G).

(H) Double labeling of SST+ (green) and CR+ (red) interneurons in ECS-preserved mouse cortex. EM images were taken from the yellow boxed region.

(I) The fluorescent labels were superimposed on the corresponding EM image taken from the yellow boxed region. The inset image demonstrated that the tissue ultrastructure was well preserved. The immunostaining work was carried out on 120 μm mouse brain sections except for the antibody penetration depth tests, which used 400 μm sections.