Name: Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies (Video) | JoVE Journal
Uploaded: 2008-01-08T13:50:00.000+00:00
Duration: 11 min 27 s
Description: Learn immunohistochemistry for protein localization in mouse epidermis using fluorescent antibodies on paraffin sections. This optimized protocol enables reliable visualization of epidermal differentiation markers.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

The complete text protocol for this experimental approach is available in <a href="http://www.springerprotocols.com/Abstract/doi/10.1385/1-59259-241-4:229">Springer Protocols</a>.

The authors have nothing to disclose.

immunohistochemistry on paraffin sections of mouse epidermis using fluorescent antibodies

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

In the epidermis. Immunohistochemistry is an efficient means of localizing specific proteins to their expression compartment. In this video, we demonstrate a reliable method for immuno localization of protein in the epidermis, which we use routinely in our laboratory.Hi, I'm Tammy from Dr.Turin's Laboratory here in the low building of the Ottawa Health Research Institute in the chronic disease program. Today we're gonna show you a procedure for immunohistochemistry using fluorescent antibodies on paraffin embedded tissue sections of mouse epidermis. In our lab, we've optimized protocols for immuno localizing epidermal differentiation markers using BO'S fixed tissue.As part of optimization, we keep the skin flat by laying it out on paper towel before fixing. This procedure involves dissection of the epidermis fixation, dehydration, paraffin embedding, and section cutting de waxing rehydration antigen unmasking antibody labeling, and finally mounting and visualization with epi fluorescence. So Let's get started.When using neonatal mice, there's No need to shave the back skin. However, when you have older mice that have fur, you need to shave the back skin away so that it doesn't get in the way of cutting the sections. When you're doing your dissection, you wanna be very careful not to put your forceps in the areas of the skin that you wanna use for your assay.So what we do is we just put the forceps around the edge of where we're going to cut, and then you lift very carefully, cut the epidermis, remembering not to touch the area that you wanna use for your experiment. When we're fixing the skin, we don't want the skin to roll up and be in the tube. We want it to be nice and flat.So what we do in order to help that is we lay the skin on a paper towel so that it stays flat while it's being fixed. So now that we have the skin nice and flat on the paper towel, what we do is we cut off the edges or we trim them in order to remove the parts that we may have damaged with the forceps. And then you cut the skin into very small pieces about two millimeter by two millimeter.Now the tissue is going to be fixed. After cutting the skin into manageable pieces, you immerse it in the boin solution. Cap the tubes, wrap the tubes with perfil and put them on a rocker.For an overnight incubation, it's important to incubate in the boin solution only 16 to 18 hours. Shorter or longer fixation periods will damage your tissue will. The incubation is over.You rinse your tissue with PBS and then through a series of ethanol washes using 30%50%and 70%alcohol. You can see that the BO is yellow in color and that as you do the washes, as the boons is diluted out, the color of the wash becomes less and less yellow. You wash two times 30 minutes each time.Following the last wash in ethanol samples are placed in a fresh 70%ethanol and may be stored at four degrees Celsius until further processing a few vents of paraffin.Embedding. Overusing frozen tissues is that tissue integrity is superior and samples can be stored for long periods of time. In general, core facilities generate more reproducible results.However, if you have the facility and a dedicated operator who has the expertise, sections can be cut in in-house. Learning to cut good sections is rather an art form. Therefore, we use a core facility.After receiving the slides back from the Core facility, you can see that the paraffin is present on the slides. As a thin film, the slides are placed on a rack wrapped with aluminum foil, just like a baked potato and incubated for five minutes at 55 degrees. After your incubation is over, quickly unwrap the slides and place the slides in toluene for three washes of five minutes.It's very important to remember that these five minute washers should be well timed, incubating too long, and toluene will not be good for your specimens. After your three washes and toluene, do a quick rinse in a hundred percent ethanol. Since ethanol and toluene form a precipitate that will interfere with your experiment, this is a very important step to remember.Next, the samples are rehydrated by incubating slides, five minutes in 95%ethanol, five minutes in, 70%ethanol, then two times five minutes in water. At this point, tissues can be processed for staining with h and e. We also use the core facility for this purpose.For antigen unmasking poor 500 mils of one time citrate buffer into a one liter plastic beaker and immerse your slides. Remember to remove the metal handle and put your beaker in the microwave. Microwave at high power for two times five minutes.Remembering to open the door to cool down the microwave in between your two incubations. After your microwaving is done, remove the slides from the boiling citrate buffer and place them into a staining dish containing warm water. We just use warm tap water and the purpose of this is to slowly cool down your slides from boiling to room temperature.Now you can wash the slides three times five minutes in PBS and we're ready to get going with immunohistochemistry. The first thing we Need to do to begin immuno staining is to dry the area around our specimens with a piece of tissue paper. In order to prevent antibody cross-contamination, we demarcate the area around our specimens with a liquid blocker.Prepare a humidified chamber by putting a filter paper into a Petri dish. Soak it with water, remove the excess water and insert your slides. The reason why we use a humidified chamber is to assure that during our incubation periods that our slides do not dry up.It's very important that the slides do not dry because drawing will increase your background. Slides are blocked at this time with incubation buffer For 30 minutes. After blocking, transfer your slides back to the slide rack and incubate in wash buffer.Remove the excess buffer just as you did before. Put the slide back in the humidified chamber and incubate with primary antibody for one hour. At room temperature.When your one hour incubation is up, put the slides back in the slide rack and wash with wash buffer for three times 10 minutes. After your washes, remove the excess wash buffer from around your specimen with a tissue paper. Put the slides back in the humidified chamber and incubate for one hour at room temperature with secondary antibody after incubating with secondary antibody, wash in three changes of wash buffer 15 minutes each.Next, we wash in three times five minutes. In PBS, remove any excess liquids surrounding the specimen and wipe the PBS away from the back of the slide to mount cover slips to the slide. We use mobile with 2.5%DAB co.In order to reduce fading, add 80 microliters of dropwise to the slides. Being careful not to introduce any bubbles. Then very slowly lower on a cover slip.Taking care not to leave any bubbles behind. Bubbles are bad because they often land right on your specimen, right where you wanna take your pictures after your mobile is dry. Now you're ready to go Look at your slides.Here is a look at a successful Staining using an FITC filter. In a 20 x objective, we see Clain six associated with all the super basal layers of the epidermis. Our lab is very interested in the expression and localization of claudins in the epidermis.With respect to tight junctions and the formation of the epidermal permeability Barrier, we've just shown you how to perform Immunohistochemistry on mouse skin. When doing this procedure, it's important to remember that you want your tissue sections to be flat and that you wanna make sure that your slides don't dry out in between incubations. So that's it.Thanks for watching and good luck with your experiments.

immunohistochemistry-on-paraffin-sections-mouse-epidermis-using

Research

JoVE Journal

Biology

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

This video demonstrates an in vivo immunofluorescence localization (IVIL) technique to study the biodistribution of tumor-targeting antibodies in a murine model of breast cancer. Upon the intravenous injection of a tumor-specific antibody in the mouse, the tumor is surgically isolated, and the localization of the antibody within the tumor tissue is confirmed via ex vivo immunostaining.

An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Here, we present a semi-automated protocol for identifying and quantifying immune and non-immune cells in skin sections using SCAnED, a free ImageJ-based macro for skin segmentation.

SCAnED - An Open-source Skin Segmentation Macro for Semi-automated Cell and Nuclei Detection in Epidermal and Dermal Skin Compartments

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Learn immunohistochemistry for protein localization in mouse epidermis using fluorescent antibodies on paraffin sections. This optimized protocol enables reliable visualization of epidermal differentiation markers.

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

Explore More Videos