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Optimization of Immunohistochemical Staining with Anti Protein

Gene Product 9,5 (PGP 9,5) Antibodies to Detecting Intraepidermal

Nerve Fiber

David Pakaya

, Yustina Andwi Ari Sumiwi

, Sri Herwiyanti

, Rina Susilowati

Department of Histology, Faculty of Medicine, Universitas Tadulako, Palu, Indonesia

Department of Histology and Cell Biology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada,

Yogyakarta, Indonesia

Keywords: Intraepidermal Nerve Fiber, PGP 9.5, Immunohistochemistry

Abstract: The intraepidermal nerve fibers (INF) are ending branches of the skin sensory nerves. These fibers can be

conceived due to the protein gene product 9.5 (PGP 9.5) as a marker recognized by immunohistochemistry.

Various studies have visualized these INF with anti-PGP 9.5 antibodies. However, this study differs in

immunohistochemical (IHC) staining methods based on the tissue thickness, the antigen retrieval process, and

the antibody product used. This study aimed to find an IHC staining optimizer with an anti-PGP 9.5 antibody

to detect INF from paraffin blocks. The INF was determined by mice skin biopsy stained with IHC anti-PGP

9.5 antibodies. This procedure was altered in the dilution, duration, incubation temperature of primary

antibodies, the tissue's thickness, and the antigen retrieval temperature. We quantitatively analyzed the

staining results. Optimization of IHC stain entail of 1:2000 dilution of the primary antibody, the thickness of

the tissues were 4 µm, overnight incubation, and low temperature of antigen retrieval. However, the results

were inconsistent. The contributing factors that enhance the IHC staining method are thinness of the tissue,

low-temperature antigen retrieval, the ratio of antibodies dilution (1: 2000), and incubation overnight at 21°C.

1 INTRODUCTION

The intraepidermal nerve fibers are the ending

branches of the skin's sensory nerves (Malik et al.,

2011). This fiber is one of the diagnostic parameters

of neuropathy (Chen et al., 2015). Neuropathy is a

clinical problem in the form of unpleasant sensations

caused by peripheral nervous system damage.

Neuropathy is assessed by decreasing the density of

intraepidermal nerve fibers, which can be identified

by immunohistochemical staining.

The data is obtained through the skin tissue

section. Therefore intraepidermal nerve fibers must

be visualized by immunohistochemical staining to

recognize the markers expressed by the nerve fibers,

one of which is protein gene product 9.5 (PGP 9.5)

(Sun et al., 2014). Various studies have visualized



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these intraepidermal nerve fibers with anti-PGP 9.5

antibodies. However, the study differs from the

method of immunohistochemical staining, the

thickness section of the tissue, the antigen retrieval

process, and the antibody product used.

Detection of intraepidermal nerve fibers can use

tissue from paraffin blocks with thin sections (<20

μm). The thin section's advantage is the penetration

of antibodies that are used faster and better and do not

require cutting tools and special microscopes. The

HRP label using to have the advantage of longer

tissue structure to observed than the fluorescent label.

A precise staining technique is needed to obtain a

quantification of intraepidermal nerve fibers. In this

study, we will optimize immunohistochemical

staining with PGP 9.5 antibody (Abcam ab8189) was

performed to detect intraepidermal nerve fibers in a

simple laboratory using tissue from paraffin blocks.

Pakaya, D., Ari Sumiwi, Y., Herwiyanti, S. and Susilowati, R.

Optimization of Immunohistochemical Staining with Anti Protein Gene Product 9,5 (PGP 9,5) Antibodies to Detecting Intraepidermal Nerve Fiber.

DOI: 10.5220/0010487400430046

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 43-46

ISBN: 978-989-758-499-2



Figure 1: Results of phase I optimization, immuno-histochemical staining feature with PGP 9.5 primary antibody on 4 μm

tissue sliced thickness. (A). 1:500 dilution. (B). 1:1000 dilution (C). 1:2000 dilution. 400x magnification.

2 MATERIAL AND METHODS

2.1 Animal Samples

This study type is a quasi-experimental study with a

cross-sectional design. It utilized two male Balb/cJ

mice strain, 8-weeks old, 20-40 g body weight. It

utilized two male Balb/cJ mice strain, 8-weeks old,

20-40 g body weight, using two mice to obtain the

skin samples by applying the principle for animal

laboratory used. This particular study has received its

permit from the ethical commission of integrated

research and development bureau (LPPT) UGM with

its certified number: 00111/04/LPPT/II/2017.

2.2 Immunohistochemical Staining

Mice were terminated, and their right foot skin was

necropted using skin punch biopsy (Premier

PMU9033505, Premier Medical, India) at 5 mm of

diameter. The resultant necropsies were grown in 5%

agarose gel and incubated overnight in

paraformaldehyde 4%; then paraffin blocks were

made. The paraffin blocks section was using

Microtome Leica RM 2235, at 4 and 15 μm thickness

with a fraction of 1/20. Immunohistochemical

staining was performed using anti-PGP 9.5 (Abcam

ab8189, Abcam, USA. MA) antibodies. The staining

was performed within several phases and

modifications upon the primary antibody

(comparison of dilution, temperature, and duration of

incubation), tissue thickness, and antigen retrieval.

2.3 Statistical Analysis

The results of the staining were analyzed qualitatively

for the intensity of the nerve fiber color.

3 RESULTS

3.1 Optimizing Immunohistochemical

Staining with Anti PGP 9.5

Antibodies

The first stage optimization is performed to obtain the

best primary antibody dilution. In the thickness of the

4 and 15 μm tissue slices using 1:500 and 1:1000

primary 1-cell antibody dilutions, the resulting

1:2000 dilution of the color with a balanced intensity

and good, but not specific. The immunohistochemical

brown color is seen in most skin epithels so that

intraepidermal nerve fibers cannot be found (Figure

1).

Phase II is carried out at 4 and 15 μ thickness and

lower retrieval antigen temperature. There was one

folded slide with 15 μm thickness. The staining

results were a picture of nerve fibers with good color

intensity, as in Figure 2. In phase III, trials were

conducted on three blocks of samples with 15 μm

thickness using the same phase II method. After the

retrieval antigen process, there were six loose slides

and six folded slides. The results of the staining look

like a picture of less good nerve fibers. In phase, IV

modification is done without antigen retrieval, a

primary antibody with 1: 2000 dilution. The results

appeared in overnight incubation as the medium-

intensity brown color, and no nerve fibers were

found. At incubation of 2 and 3 nights, the results

appear in brown with a concentrated intensity and are

non-specific.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )



Figure 2: Intraepidermal nerve fibers with anti-PGP 9.5

antibodies are shown as brown perpendicular lines that pass

through the basal membrane (indicated by arrows) with

1000x magnification.

Phase V was performed at 15 μm thickness,

dilution of primary antibody at 1: 2000 with

modification of the antigen retrieval temperature and

the primary antibody's incubation temperature. The

result showed several loose or folded slides, and the

staining results appear as a brown stain with moderate

intensity, and no nerve fibers are found. Stage VI was

performed at a thickness of 4 μm, with 1: 2000

dilution of primary antibody and the modification of

lower retrieval antigen temperature. The result

showed no slide folding or losing, and there was a

picture of nerve fibers.

4 DISCUSSION

Optimization is important to make a good quality of

tissue staining. We will optimize

immunohistochemical staining with PGP 9.5

antibody to detect intraepidermal nerve fibers using

skin tissue from paraffin blocks to diagnose

neuropathy. The immunohistochemical optimizing at

phase I, a 1:2000 dilution was obtained with its best

stain production and balanced and good intensity. In

phase II, the nerve fibers appear well at 15 μm

thickness. In another study, nerve fibers appeared

well on 5 μm slice thickness of paraffin blocks

(Thomsen et al., 2009; Ventura et al., 2011), 50-100

μm thicknesses with frozen section (Stavniichuk et

al., 2011) and 2 μm thickness with a confocal

microscope (Periquet et al., 1999). To quantify the

number of nerve fibers with specific antibody

markers, it's is recommended to use thin slices

(Beiswenger et al., 2008). In phase III, the same

thickness and method do not produce a consistent

picture. This phenomenon is influenced by other

processes such as antigen retrieval temperature and

the incubation of primary antibodies.

Good nerve fibers appear after antigen retrieval

was conducted with low temperatures. However,

these low-temperature modifications remain

inconsistent, just like the results in phase V. In the

case of axon injury, the image of nerve fibers is also

obtained after low-temperature antigen retrieval

(Stone et al., 2009). This study's obstacles were the

thickness of the skin foot sample (15 μm) with the

cornification at risk of loose or folded after antigen

retrieval. In phase IV, the retrieval antigen process is

not performed, but the results are not specific. The

phase VI optimization was carried out at 4 μm

thickness with the same retrieval antigen temperature

as stage V, obtained a picture of nerve fibers with

balanced color intensity. To get a good picture of the

nerve fibers needs to be preceded by the retrieval

antigen at low temperatures. The open epitope will be

very good to bind to the incubated antibody. A good

description of the nerve fibers was obtained in the

second stage of optimization with 15 μm slice

thickness and stage VI with 4 μm slice thickness. The

thickness of 4 μm slices is the most likely to be done

because at 15 μm, the thickness is risky to lose or fold

after retrieval antigen.

To quantitate the intraepidermal nerve fibers

density, the stereology principle was used. The

samples were randomly and systematically obtained

from the paraffin block slices that have the same

thickness. With this method, we will get some slices

to be quantification according to the specified

fraction. The entire cross-section must look good to

be quantified, so when using a 15 μm thickness, it will

have difficulty reporting the results. Skin tissue is not

isotropic, i.e., uniformity in parameter values in all

directions, so it takes orientation or determination of

the direction of network cutting to avoid bias (Witgen

et al., 2006). However, manual quantification still

possesses the possibility of bias, so that software

usage is recommended. The software will generate

randomly oriented virtual isotropic fields in a 3D

virtual field containing parallel lines so that any nerve

fibers tangent to the line can be quantified (Karlsson

et al., 2013; Karlsson et al., 2016). Inconsistent

optimization results and the shortcoming of manual

quantification may cause difficulty in conducting the

study.

Optimization of Immunohistochemical Staining with Anti Protein Gene Product 9,5 (PGP 9,5) Antibodies to Detecting Intraepidermal

Nerve Fiber



5 CONCLUSION

The immunohistochemical staining method with the

most optimal anti-PGP 9.5 antibodies was performed

on thin, with low-temperature antigen retrieval and 1:

2000 antibody dilution incubated overnight at 21°C.

The results of this study can be directed to become

a diagnostic method of neuropathy.

ACKNOWLEDGEMENTS

The author would like to express gratitude to BPPDN

Dikti 2015 (906.II/E4.4/2015). Mrs. Wiwiet

Setyowati, Histology, and Cell Biology Laboratory of

UGM Medical School.

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JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )