Protocol
A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry

https://doi.org/10.1016/j.brainresprot.2005.02.004Get rights and content

Abstract

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

Section snippets

Type of research

  • (i)

    Immunohistochemical detection of β-galactosidase (β-gal) as a reflection of neuronal activation in transgenic mice containing a tau-lacZ fusion gene regulated by the promoter for c-fos [17].

  • (ii)

    Measurement of c-fos protein production as a reflection of metabolic activation of neurons [4], [15].

  • (iii)

    Study of c-fos activation as a reflection of neuronal activation patterns in kindling induced seizures [16].

  • (iv)

    Use of the brightness-area-product (BAP) as a measure of the amount of high-contrast material in a

Time required

The kindling procedure from electrode implantation to the time of sacrifice after electrical stimulation requires 7–8 days. Preparation time of tissue including freezing, sectioning, mounting is 4 h per mouse.

Immunohistochemical staining requires 28 h including fixation of slide mounted sections, serum blocking, primary and secondary application steps and coverslipping based upon a 56-slide run. One day is then allowed for mounting media to dry before slide cleaning.

Quantitative analysis of

Animals

Twenty fos-tau-lacZ (FTL) transgenic mice were obtained from the Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Australia [17]. Generation and genotyping of these mice was as described previously [17]. Adult FTL mice aged 8–12 weeks were kept on a standard laboratory diet with access to food and water ad libitum and exposed to a 12-h light/dark cycle commencing light at 7:00 am in a vivarium maintained at 23 °C room temperature. All studies were performed with the

Kindling

All mice were anaesthetised with chloral hydrate 4% w/v at a dose of 10 ml/kg bodyweight and mounted in a stereotactic frame (Kopf™, Tujunga, CA, USA). A bipolar twisted, polyimide-coated, stainless-steel stimulating/recording electrode (Model MS303/1; Plastics One, Roanoke, VA) was then implanted into the basolateral nucleus of the left amygdala using coordinates derived from the Paxinos and Franklin atlas [12] (coordinates: 1.9 mm caudal to bregma: 3.1 mm lateral to bregma, 4.6 mm ventral to

Results

We found good correlation between individual blinded observer interpretation of qualitative β-gal expression intensity by immunofluorescence as scored by a 0–3 scale and more quantitative interpretation of brightness by the protocol presented as is depicted in Fig. 2. The degree of correlation between the two scales was calculated statistically for each subregion across all animals in each of the experimental subgroups. As depicted in Table 1, the correlation, as calculated for each subregion

Discussion

This paper describes a means to quantitatively assess the brightness of a single channel immunofluorescent histological image and infer the relative abundance of an epitope using commercially available software. In the presented example, we have deliberately examined an epitope expressed in neuronal cytoplasm and processes rather than nuclei to emphasize the utility of such a technique. Where an antigen is expressed in nuclei alone an estimate of abundance can be gained by counting positive

The apparent appropriate threshold varies between subregions

Where heterogeneous regions are being examined in the one experimental run, particularly regions differing in terms of white matter content or background vasculature density, markedly differing levels of background brightness may be encountered which may not be clear until digital analysis. It is therefore important in these settings to determine necessary thresholds for each area individually.

A small but definite BAP is generated in areas which seem uninvolved to the eye after thresholding

Secondary antibodies that tend to form precipitates may cause this phenomenon. For abundant antigens,

Quick procedure

(i) Tissue preparation

  • Sacrifice of animals at designated timepoint.

  • Freezing of brain over isopentane/dry ice slurry and cryosectioning.

  • Serial sections mounted on AES slides.

(ii) Immunohistochemistry

  • Fixation in PFA.

  • Washes in PBS.

  • Wax pen demarcation around sections, including multiple sections with no primary antibody exposure controls.

  • Block with 10% donkey serum.

  • Incubate with rabbit anti-β-gal primary antibody.

  • Incubate with donkey anti-rabbit Alexa-Fluor 488 conjugated secondary antibody.

Acknowledgments

We thank Sanofi-Synthelabo™ for donation of a stereotaxic frame for surgery and Compumedics™ for supplying EEG monitoring equipment. Financial support for Dr. Paul D Smith through a research scholarship from the Royal Australasian College of Surgeons and the National Health and Medical Research Council (NHMRC) and technical assistance from Mr. Lucas Litewka are also gratefully acknowledged.

References (17)

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