This procedure begins upon receipt of a 4%PFA perfused, externalized, and cryo protected brain, which has been sent to Neuroscience associates for embedding using alignment landmarks embedded in the surrounding matrix. The brain can be sectioned at the coronal plane to produce serial sections at a desired thickness, which will represent the entire anterior posterior extent of the brain using specialized baskets and containers. Batch immuno processing can be performed in order to determine the spatial expression pattern of a protein throughout the entire brain.
Hi, I'm Dr.Shaheen Zur from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hi, I'm Dr.Mark Burke, also from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hello, I'm Dr.Mo Petto, director of the Visual Neuroscience Laboratory at the School of Optometry, university of Montreal.
Today we'll show you a procedure for batch immuno staining for large scale protein detection in a whole monkey brain. We use this procedure in our laboratory to study the expression patterns of target proteins across the whole brain and to compare and contrast the expression of those proteins within the same brain. So let's get started.
Prior to histological processing, obtain a primate brain, which has been preserved via systemic perfusion and post fixation and cryo protected using graded sucrose solutions. At this stage, the brain is sent to neuroscience associates to be embedded and free sectioned using their proprietary technology. Upon return, one will observe that seven alignment landmarks are placed in the embedding matrix so that sections can be oriented properly and realigned after histological processing is complete.
While sectioning is performed, digital images are taken that can later be used for 3D reconstruction of anatomical maps. Neuroscientist associates processes our tissue to yield serial 50 micrometer thick free floating coronal sections over the entire brain. Once complete, we can begin histological processing of the sections.
When we begin histological processing, we typically have around 140 sections to handle per antibody or stain. Here we will demonstrate the technique with a smaller sample of sections treated with the antibody SMI 32, which is a monoclonal antibody against non phosphorylated neurofilament proteins. Throughout the procedure, specialized staining dishes and baskets are used, allowing for a large number of free floating sections to be processed simultaneously and efficiently.
This ensures that the resulting stain is consistent across all sections. The first step is to incubate the sections in a PBS based solution containing TRITTON X 100 and normal horse serum for 60 minutes. This will reduce the nonspecific binding of antibody molecules that results in background staining.
Next, the sections are incubated overnight. In A PBS based SMI 32 antibody solution, supplemented with tritton X 100 and normal horse serum the following day, the sections are washed for three 10 minute periods in washing solution and then incubated for two hours at room temperature. In a PBS based biotinylated Muse secondary antibody solution containing Triton X 100 and normal horse serum.
After a set of three additional 10 minute washes, the sections are placed in a solution of avadon biotin conjugated horse radish peroxidase complex for one hour at room temperature. Finally, after another set of washes, the sections are placed in a D amino benzine reaction for 10 minutes, which produces a brown stain within the immunoreactive neurons. The reaction is then stopped by removing the staining basket and immersing the sections in PBS.
After two to three, five minute rinses in PBS sections are ready to be mounted individually on glass slides to begin the mounting process using a large Petri dish and very soft paint brushes. Sections are lifted from the PBS buffer container and individually mounted onto gelatin coated glass slides. The samples are then allowed to air dry overnight in a fume hood the following day.
The sections are rinsed by dipping them in di deionized water to wash off any salt crystals that may be left over from the mounting process. The slides are then dehydrated in graded ethanol steps cleaned in xylene and cover slipped with per mount mounting medium. At this stage, slides should be stored for about a week to 10 days in a horizontal position to allow the mounting medium to harden.
After this, the slides can be stored in a slide box or subjected to imaging via microscopy. This method produces a complete expression profile of a target protein of interest across an entire monkey brain. Here we show representative coronal sections that provide a snapshot of FMRP, new n and SMI 32 expression.
In the same monkey brain. We've just shown you how to complete batch immuno staining for a target protein of interest across the whole brain. When doing this procedure, it's important to ensure that all sections undergo mild agitation while being incubated in various solutions, and that at all times they remain completely immersed.
In addition, take your time while glass mounting the dissections and remove as many of the wrinkles and fold as possible without damaging the integrity of the tissue. So That's it. Thanks for watching and good luck with your experiments.
