Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

The overall goal of this procedure is to bypass the limitations of indirect immunofluorescence to study neural progenitor cells. When suitable primary antibodies from different host species are unavailable. Initially, a thymidine analog is injected to label the dividing cells, after which the tissue is harvested, processed, and sliced into cryo sections.

Then sequential multiple immunofluorescence is performed to begin. An unlabeled primary antibody is applied, which is then bound by a fluorescently labeled secondary antibody. Next, the open pericopes on the first secondary antibody are blocked with serum from the primary antibody species, and all the immunoglobulins that could be recognized by the second secondary antibody are covered with unconjugated fab fragments.

This allows for the tissue to be treated with a second primary antibody, which can be labeled by a different secondary anti antibody. Ultimately, images of the sections are used to study the development and regulation of neural progenitor cells in response to specific stimuli and their involvement in particular brain functions. The main advantage of this technique over existing methods like simultaneous multiple immunofluorescence, is that it circumvents problems usually arising from the need of using primary antibodies raised in the same host species.

This method can help answering key questions in the field of adult neurogenesis, such as the proliferation of hippocampal progenitor subsets in response to physiological or pharmacological stimuli, brain pathologies or aging. Generally, individuals new to this method will struggle because the multiple labeling with primary antibodies from the same host species might lead to undesired antibody cross activities without adequate blocking. First, weigh the animals to be injected and prepare a fresh 10 milligram per milliliter thymidine analog stock solution for the injection.

When the solution is ready, prepare a weight adjusted dose in a fine dosage syringe with a 30 gauge needle. Then restrain the mouse by the scruff and inject the thymidine analog intraperitoneal at the desired time point deeply anesthetize the mouse and trans cardio. Perfuse it with 10 milliliters of ice cold PBS, followed by 40 milliliters of fresh ice cold formaldehyde solution.

Then dissect out the brain and post, fix it in the same fixative following the fixation. Incubate the collected brains for 24 hours in 10%sucrose at four degrees Celsius. The next day, transfer brains into 30%sucrose and incubate for another 48 hours.

With the brains ready for cryo protection, freeze them in negative 25 degrees Celsius isop pentane. Later, use a cryostat to cut 40 micron coronal sections for long-term storage. Transfer the sections to an antifreeze solution and keep them at negative 20 degrees Celsius.

Begin with transferring the sections from the antifreeze solution to TBS with the help of a fine brush. Rinse the sections thoroughly, starting with an overnight wash at four degrees Celsius, followed by five 10 minute washes at room temperature. All these incubations should be performed with continuous gentle agitation.

If one of the antigens of interest is a thymidine analog, a hydrochloric acid denaturation step has to be implemented at this point, followed by a neutralization step in bore buffer. Proceed with permeable and blocking of the unspecific antibody binding sites in TBS plus. Let this incubation go for one hour at room temperature, then incubate in the first primary antibody diluted in TBS plus overnight at four degrees Celsius the next day.

Wash the sections three times in TBS and once in TBST. Let each bath go for 10 minutes. Then incubate in first fluorochrome conjugated secondary antibody for three hours at room temperature.

From now on, protect the sections from light exposure. After another wash series, transfer the sections into a 10%host species serum solution from which the two primary antibodies were made. Incubate the sections in this solution for three hours to saturate the open pericopes on the first secondary antibody.

Later rinse three times in TBS and once in TBST to remove the serum. Then prepare a solution of TBS plus with 50 micrograms per milliliter of unconjugated monovalent fab fragments directed against the host species of the primary antibody. This covers the epitopes that could otherwise be recognized by the second secondary antibody.

Transfer the sections into this solution and incubate overnight at four degrees Celsius. After the incubation, rinse the sections at least three times in TBS and once in TBST. During transfers, swab the mesh inserts containing the sections on a paper towel to remove residual fab leavings.

Now incubate the sections in the second primary antibody overnight at four degrees Celsius. After another wash series, apply the second secondary antibody for three hours at room temperature. Finally wash the sections three times in TBS and proceed with mounting the sections gelatin.

Air dry the slides and cover slip them using an ANTIFA mounting medium image. The fluorescence labeled sections with a confocal laser scanning microscope. Take image stacks at random positions along the entire extent of the dentate gyrus at 400 fold magnification.

Analyze at least 50 randomly selected cells of interest per hemisphere for co labeling of the different markers. To calculate absolute numbers for specific newborn cell populations, multiply the percentages of co labeled cells with the total numbers of newborn cells as estimated from DAB staining analysis. The described methods were used to quantify and characterize newborn cells in the postnatal and adult hippocampus of a neurogenesis deficient cycling.

D two knockout mouse model, DAB staining against different proliferation markers like KI 67 or BRDU consistently revealed differences in newborn cell numbers between wild type and knockout mice. BRDU labeled newborn cells could be phenotyped with a conventional multiple immunofluorescence protocol of simultaneously applying primary antibodies against B-R-D-U-G-F-A-P nest in GFP and double courtin. This allowed successful identification of newborn type one, type two A, type two B and type three progenitor cells within a single specimen.

Alternatively, with longer time intervals between BRDU injection and sacrifice of the animal, the final numbers of newborn neurons could be analyzed by simultaneous BRDU new n immuno staining. The sequential immuno labeling protocol worked perfectly if sections were first incubated simultaneously with rabbit anti KI 67, goat anti GFP, and Guinea pig anti double cordon antibodies, and secondly, with rabbit anti GFAP antibody, but not vice versa. After watching this video, you should have a good understanding of how to visualize up to four different antigens in the single tissue section using primary antibodies raised in the same host.

While attempting this procedure, it's important to remember to include appropriate controls to eliminate the possibility of antibody, cross reactivity or false positives. Be aware that the sequence of primary antibody incubations might strongly influence the outcome of the protocol.