Conventional Staining & Coverslipping

Cell Body Staining

Cell body staining is performed using the Tissue-Tek Prisma, Automated Slide Stainer (Sakura). Staining is performed in groups of 60 slides. A Nissl (thionin) staining protocol is used. Staining times were standardized for all samples. All reagents are routinely tested for lot-to-lot consistency.

Dehydration

Fluorescent samples that have undergone immunofluorescence or similar enhancement are dehydrated using the Tissue-Tek Prisma system. Dehydration is performed by passing the slides through a linear ethanol gradient (70%→95%→100%) for several minutes for each step. The slides are then coverslipped.

Giemsa Counterstain

For slides that have undergone enzyme histochemical processing (HC), a Giemsa based counterstain is performed, followed by dehydration of the slides and coverslipping. Giemsa is a cell body stain and also acts to enhance the HC label.

Coverslipping

Slides are coverslipped using the Tissue-Tek Glas g2, Automated Slide Coverslipper (Sakura). A xylene-based mounting medium is used for all samples. After coverslipping, slides are manually unloaded onto a drying rack and examined to ensure that the coverslip does not overhang the edges and that mounting media covers all sections. Slides are allowed to dry for 24 hours before further use.

Enzyme and Immuno-Histochemical Staining:

Both enzyme histochemistry (HC) and immunohistochemistry (IHC) are performed on the slide. Staining is performed using a modified LabVision720 (Thermo Scientific) or two parallel Dako Autostainers. Modifications were made to the slide holders of both instruments to facilitate slide-to-slide consistency. A closed-system, forced air, steam humidifier is also connected to the chamber of each unit, and this provides important direct humidity control of the staining chamber.

IHC Detection of Cholera Toxin Subunit B (CTB)

For CTB, the detection protocol consists of a short protein block, prolonged incubation in primary antibody, incubation in secondary antibody, incubation in Avidin-Biotinylated solution (Vector “ABC” Kit), and finally through a DAB histochemical reaction. Slides are hydrated in PBS prior to staining and washed with PBS between each incubation step (with the exception of protein block →1º Antibody).

HC Detection of Biotinylated Dextran Amines (BDA)

BDA is already conjugated with biotin, therefore the detection protocol calls for a prolonged incubation in Avidin-Biotinylated solution (Vector “ABC” Kit), followed by a final DAB histochemical reaction.

Diaminobenzidine (DAB) Histochemical Reaction

The principal form of DAB (Thermo Scientific) used throughout the project is heavy metal enhanced DAB. The solution is prepared fresh, with H2O2 added no more than 5 minutes prior to immersion of the slides. Following the incubation, slides are thoroughly washed with PBS to stop the reaction. For some early datasets, an alternative version of DAB was used (Invitrogen).