Journal of Histochemistry and Cytochemistry, Vol. 47, 1465-1470, November 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Antigen Retrieval in Prion Protein Immunohistochemistry

Bart Van Everbroecka, Philippe Palsa,b, Jean-Jacques Martina,c, and Patrick Crasa,c
a Departments of Neurobiology, Born Bunge Foundation, Antwerp University, Wilrijk, Belgium
b Molecular Genetics, Born Bunge Foundation, Antwerp University, Wilrijk, Belgium
c Neuropathology, Born Bunge Foundation, Antwerp University, Wilrijk, Belgium

Correspondence to: Patrick Cras, Born Bunge Foundation, Laboratory of Neurobiology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Transmissible spongiform encephalopathies are a group of neurodegenerative diseases occurring in both humans and animals and are most likely caused by prions. Neuropathological confirmation of the clinical diagnosis has been a problem because of the difficulty in epitope retrieval from formalin-fixed, paraffin-embedded brain specimens. Many different protocols for the detection of prions in brain tissue have been used. Thus far, picric and/or formic acid, steam autoclaving at 121C of sections, microwave treatment, and 4 M guanidine thiocyanate treatment have been suggested. The objective of our experiment was to obtain the standard pretreatment(s) resulting in optimal immunostaining. In the experiment, successive tissue slides of brain specimens of several Creutzfeldt–Jakob disease and control patients were stained using different combinations of pretreatments. Using densitometric analysis, several well-defined locations per section were examined and prion immunostaining was quantified. The results showed that autoclaving is necessary for antigen retrieval and cannot be substituted by microwave treatment. The best results were obtained when the following combination was used in the specified order: 15 min saturated picric acid, 10 min steam autoclaving at 121C, 5 min 88% formic acid, and 2 hr 4 M guanidine thiocyanate at 4C. (J Histochem Cytochem 47:1465–1470, 1999)

Key Words: prion protein, immunohistochemistry, transmissible, spongiform, encephalopathy, Creutzfeldt–Jakob, antigen, neuropathology


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases characterized by a rapidly progressive deterioration (in cognitive function and/or coordination) which always leads to death (Parchi and Gambetti 1995 ). TSEs occur in humans and in animals. The most likely cause of the TSEs is the prion protein form designated PrPSc, named after scrapie, the oldest known form of prion disease, which originated in sheep and goats (Prusiner 1992 ). How prions cause brain damage is unclear at present, but all hypotheses suggest that posttranslational modification of the native prion protein (PrPC) by PrPSc to form amyloid fibrils is a central event in pathogenesis (Prusiner 1995 ).

In humans, Creutzfeldt–Jakob disease (CJD) is the most widespread TSE (incidence 1/million/year). Clinically, patients can be diagnosed as possible or probable CJD patients but neuropathological conformation is necessary to obtain a definite diagnosis. Neuropathological investigation is based on a triad of histological lesions: spongiosis, neuron loss, and reactive astrogliosis (Budka et al. 1997 ).

After the discovery that the prion protein is linked to TSE and CJD, many protocols for immunohistochemical (IHC) detection of prion depositions in brain tissue were designed. The main problem was the difficult epitope retrieval after formalin fixation and paraffin embedding of brain tissue. To increase the sensitivity of detection, several pretreatments were suggested by different research groups, i.e., picric acid, formic acid, steam autoclaving at 121C (Kitamoto et al. 1985 ), microwave treatment, and guanidine thiocyanate (Haywood et al. 1994 ; Goodbrand et al. 1995 ; Bell et al. 1997 ).

The objective of our study was to determine which single pretreatment or combination of pretreatments gave the best result for IHC analysis of the prion protein in brain specimens of CJD patients. Sections from the cerebellum and occipital cortex from five different definite CJD patients and two control subjects were selected. Successive tissue slides of these sections were stained with a total of 17 combinations, ranging from no to four pretreatments. To make the results independent of the detected epitope, two different monoclonal antibodies (MAbs) were used in the experiment. To ensure that environmental or experimental events could not influence our conclusions, a number of experimental guidelines were designed. Adequate areas in which to compare the different pretreatments were selected by visual examination. The selection protocol focused on the synaptic staining pattern because this is the most common in CJD and the most difficult to detect. In a second stage, other patterns of prion deposition (e.g., kuru plaques and perivacuolar deposits) were evaluated to detect differences in the enhancement of antigen retrieval. The immunostaining was quantified by digital densitometry. The results were expressed as relative density to the optimal staining pretreatment in that specific location.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

To study epitope retrieval, five definite CJD patients and two control patients were selected. The clinical diagnosis of sporadic CJD was confirmed in all five cases by neuropathological investigation. IHC for the prion protein was performed, using hydrated autoclaving and formic acid as pretreatments. In two patients, intense prion immunostaining was found, two cases showed moderate immunostaining, and in one patient poor immunostaining was observed. Kuru plaques were present in one patient; perivacuolar staining and synaptic staining were present in all patients. The first control patient was a neuropathologically confirmed case of Alzheimer's disease. The second control patient suffered from breast cancer. In all patients except for one CJD patient (occipital cortex was not available), both the occipital cortex and the cerebellum were studied.

All tissue was treated with 98% formic acid for 1 hr to reduce infectivity and embedded in paraffin. Six-µm tissue sections were made on a microtome using a separate knife and safety gloves. Tissue sections were picked up on 0.1% poly-L-lysine (Sigma; St Louis, MO)- treated glass slides. The sections tended to come off when Superfrost Plus glass slides were used in staining procedures with multiple pretreatments (not shown).

To ensure that the immunostaining among the different combinations of pretreatments was comparable, a number of guidelines for the experiment were established. Serial tissue sections were made and numbered for use. All combinations of pretreatments were examined in one experiment, so identical reagents, incubation times, and environmental conditions were used for all pretreatments. The final guideline was that sections were placed in H2O, as substitute, when other sections underwent a pretreatment.

The sequence and reagents for the pretreatments were based on the United Kingdom consensus protocol for prion protein detection (Bell et al. 1997 ).

Picric Acid (PA)
Tissue slides were incubated with 250 µl of saturated picric acid (5%) (Vel; Leuven, Belgium) for 15 min at room temperature (RT) and were subjected to multiple tapwater washes. Safety guidelines were supplied by Vel. PA is both toxic and explosive. Safety guidelines R 1, 4, 23/24/25 and S 35, 36/37, 45 must be used when working with PA.

Hydrated Steam Autoclaving (HA)
Tissue slides were autoclaved (Microclaaf; Selecta, Abrera, Spain) for 10 min at 121C using 10 mM citric acid (Merck; Darmstadt, Germany), pH 6, as recovery buffer. Afterwards the sections were allowed to cool and finally washed in distilled water.

Microwave Treatment (MT)
Tissue slides were microwaved (M404 Whirlpool; Philips, Eindhoven, The Netherlands) twice for 5 min using 10 mM citric acid (Merck), pH 6, as recovery buffer. Afterwards the sections were allowed to cool and finally washed in distilled water.

Formic Acid (FA)
Tissue slides were treated with 88% formic acid (Merck) for 5 min and were washed in distilled water.

Guanidine Thiocyanate (GdSCN)
Guanidine thiocyanate 4 M (Merck) was precooled at 4C. The GdSCN solution was pipetted onto the tissue slides and they were incubated for 2 hr at 4C. After incubation, the GdSCN solution was removed with adsorbent paper which was disposed of as biohazardous material.

The IHC protocol used in the experiments was as follows. First, the tissue was rehydrated and deparaffinized. Second, the PA treatment was completed and was followed by endogenous peroxidase blocking by treatment for 30 min in methanol containing 0.3% H2O2. Then the slides were treated by HA or by MT. The next step was the FA treatment. The GdSCN treatment was the last pretreatment.

Tissue sections were then exposed to 1:25 normal swine serum (NSS) in TBSB [150 mM NaCl, 1% BSA (Sigma), 50 mM Tris-HCl, pH 7.4] for 30 min to block nonspecific binding sites. Sections were incubated overnight with primary antibody at RT in humid chambers. Two anti-prion MAbs were used: 3F4 (Senetek; St Louis, MO) diluted 1:2000 and F89/160.1.5. This last was a gift from Dr. K. O'Rourke (Agricultural Research Service, US Department of Agriculture, Washington, DC). This antibody was used at a concentration of 55 ng/ml. The epitope of the antibody is IHFG, which is conserved in most species. To detect MAb binding, we used the avidin–biotin complex method (ABC kit; Amersham, Poole, UK). Bound antibody was detected by incubation with secondary antibody (biotinylated goat anti-mouse IgG 1:100 in TBSB, 30-min incubation) and avidin–biotin–horseradish peroxidase complex (1:200 in TBSB, 1-hr incubation). As staining mixture, 0.05% 3.3' diaminobenzidine tetrahydrochloride (DAB; Jansen Chimica, Olen, Belgium) in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.6) with 0.002% H2O2 was used for 5 min. After extensive washing, the sections were counterstained with Harris' hematoxylin for 30 s. Then the sections were dehydrated and mounted with Eukitt (Labonord; Mönchengladbach, Germany).

Immunostaining was investigated by visual examination and quantified by digital densitometry. Appropriate areas for quantification were chosen by visual examination of all sections. Three to four areas per brain region were selected, using the HA-pretreatment sections as baseline reference for the presence of synaptic immunostaining. These selected areas were then carefully indicated on the sections of the same brain location for all pretreatments. This resulted in 43 different areas to quantify for synaptic staining and 19 for other staining patterns. Using an LCD camera, images of these areas were digitized. The image analysis software quantified the immunostaining in the selected areas. Optical density was measured by the software per pixel. The darker the staining in one pixel, the higher the value (D/pix), between 0 (no staining) and 3 (dark brown staining) that was given to that pixel. The final density is the sum of all the separate D/pix (integrated density). The highest density measurement was then set to 100% and relative density of immunostaining of the other pretreatments in the same location, referred to as %RD, was calculated. This calculation resulted in a data collection that was independent of the patient and the brain location used.

It was on this set of calculated data that the statistical analysis was performed. The Mann–Whitney U rank-sum test was selected for this analysis. This test is the most rigorous nonparametric test, which allowed correct analysis of small numbers and not normal distributed data.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

No Pretreatment
Microscopic examination revealed that very few prion depositions were visible. Immunostaining was absent in 75% of all areas examined. Only in the two cases with previously found intense prion depositions, immunostaining was faintly but consistently present (Figure 1E). The digital densitometry had the same result: in these tissue sections 5% ± 3%RD was detected (Figure 2). In the following paragraph this measurement was used as reference value to determine the effect of the pretreatments.



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Figure 1. Immunohistochemistry of the prion protein with the 3F4 MAb. (A–D) the dentate nucleus (DN) of the same CJD patient (x158). Maximal staining, obtained after all pretreatments were used, results in widespread staining of the prion deposits in the DN (A). When only autoclaving is used, the number of reactive sites and the intensity of reactivity are significantly reduced (B). When microwaving was used in combination with acid treatments and guanidine thiocyanate, only faint reactivity was observed (C). When formic acid was used, no staining was observed in the DN (D). In the granular layer of the same CJD patient, on the other hand, some staining was present even when no pretreatments were used (E; x158). In the DN of a control patient with Alzheimer's disease no reactive deposits were observed even after all pretreatments were used (F; x158).



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Figure 2. Comparison of relative immunodensity (%RD) after single pretreatment. No pretreatment displayed 5 ± 3%RD, the least immunodensity (A). Formic acid showed 15 ± 8%RD (B), picric acid 11 ± 5%RD (C), and guanidine thiocyanate 13 ± 5%RD (D), a significant increase (p<=0.03). Autoclaving (E) resulted in 41 ± 5%RD, which was the highest increase in immunodensity for one pretreatment (p=0.0001).

In the control sections no immunoreactivity was visible (Figure 1F). The digital densitometry did recognize some residual background. This was the result of the counterstaining procedure applied. When pretreatments were used on these slides, no increase in background was observed, as expected. The pooled data of the CJD patients were used as reference value to calculate the relative density of the background. This result showed an immunodensity of 2% ± 3%RD (Figure 2A). This differs significantly from the value found after no pretreatment (p=0.006).

Single Pretreatment
The result of MT treatment (Figure 4) showed an immunodensity of 6% ± 4%RD. There was no difference with the result when no pretreatment was used.



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Figure 3. Comparison of relative immunodensity (%RD) after multiple pretreatments. The value of autoclaving (HA) is displayed (empty bar, A) as reference value. Combination of HA with picric acid (41 ± 10%RD, B) or guanidine thiocyanate (46 ± 10%RD, C) did not lead to a significant increase in immunodensity. The combination of HA and formic acid (FA) displayed 51 ± 10%RD (E) and the combination of HA with picric acid and guanidine thiocyanate showed 57 ± 5%RD (D), which were significant increases in immunodensity (p<=0.02). Immunodensity was further enhanced by combining HA, FA, and guanidine thiocyanate (82 ± 11%RD, F) and the combination of HA, FA, and picric acid (85 ± 10%RD, G) (p<=0.01. The best result was always obtained by the combination of all four pre-treatments (100%RD, H).



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Figure 4. The effect of microwaving (MT) (open bar) or the combination of microwaving with acid and guanidine thiocyanate pretreatments (MT + all) (striped bar) on prion immunostaining is displayed. Synaptic staining after MT resulted in 6 ± 4%RD (A) and after MT + all in 18 ± 5%RD (B). This indicates that microwaving did not increase immunodensity compared to the same pretreatment(s) without microwaving. Other types of staining (kuru plaques, perivacuolar staining) showed a significant increase of immunodensity (p<=0.012). Microwaving alone resulted in 35 ± 6%RD (C). Microwaving combined with all pretreatments resulted in 87 ± 6%RD (D).

After the use of FA, PA, and GdSCN, microscopic examination showed improvement. Only 25% of all areas showed no immunostaining in the CJD patients with mild and moderate prion deposits (Figure 1D). When HA was used, immunostaining was found in all areas (Figure 1B).

Tissue sections incubated with FA showed an immunodensity of 15% ± 8%RD (Figure 2B). Sections treated with PA displayed an immunodensity of 11% ± 5%RD (Figure 2C) and after treatment with GdSCN an immunodensity of 13% ± 5%RD (Figure 2D) was found. All these treatments increased immunostaining 2.5-fold (p<0.03).

Tissue sections that had been treated with the HA displayed an immunodensity of 41% ± 5%RD, which was an eightfold (p=0.0001) increase of immunostaining (Figure 2E).

Multiple Pretreatments
Visual examination revealed HA to be the necessary pretreatment. No combination of pretreatments increased immunostaining to the same magnitude as autoclaving. It was also observed that in patients with intense immunostaining the number of deposits did not substantially increase when HA was combined with other pretreatments; only the intensity increased strongly. In the other patients, a strong increase both in reactive sites and in intensity was observed.

The digital analysis gave the following results. (a) The combination of HA with PA or GdSCN did not result in a significant increase compared with the HA pretreatment (Figure 3B and Figure 3C). The combination of FA with PA and/or GdSCN also did not reveal a significant increase compared with the FA pretreatment (not shown). (b) The combination of FA or PA and GdSCN with HA did result in a significant 1.25-fold increase compared with the HA pretreatment (p=0.02). For the combination of HA + FA, a relative density of 51% ± 10%RD was calculated (Figure 3E). If HA + PA + GdSCN were combined, the resulting immunodensity was 57% ± 5%RD (Figure 3D). (c) The combination of FA and HA with PA or GdSCN resulted in a 1.52-fold increase compared to the FA + HA combination (p=0.01). The calculated immunodensity for the combination HA + FA + PA was 85% ± 10%RD (Figure 3F) and for the combination HA + FA + GdSCN was 82% ± 11%RD (Figure 3G). (d) The best result was always obtained with the combination of all four pretreatments (Figure 1A), which gave an additional 1.21-fold (p=0.02) increase over the previously described combinations (Figure 3H).

Other Staining Patterns
In brain areas with other deposition types, MT treatment (Figure 4) showed an immunodensity of 35% ± 6%RD, which was a sevenfold (p=0.01) increase in immunostaining (Figure 4C). This result is clearly different from that of the effect of MT treatment on synaptic staining (Figure 4A). This effect was also seen when microwave treatment was combined with other pretreatments. In combination with FA, PA, and GdSCN, the synaptic staining had a relative immunodensity of 18% ± 5%RD (Figure 4B) whereas the other staining patterns had a relative immunodensity of 87% ± 6%RD (Figure 4D).

This effect was not observed with other pretreatments (not shown).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The results showed that hydrated autoclaving was the single pretreatment that resulted in a maximal increase in immunostaining (eightfold increase compared to no pretreatment). If hydrated autoclaving was combined with formic acid, the relative density was further improved by 40%. If picric acid and guanidine thiocyanate were supplemented in the described sequence to the above combination, the best immunostaining was obtained.

For the neuropathological diagnosis these results are important. If no pretreatments are used, there is a high risk of false-negative immunostaining. If, on the other hand, the complete sequence of PA, HA, PA, and GdSCN is used, the chance of false-negative immunostaining is highly reduced. After completion of the experiment, we have used the full sequence protocol to determine prion deposition in a retrospective study of 79 CJD patients and adequate neurological controls. In this study we were able to detect prion depositions in all CJD patients but none in the controls (unpublished observations).

When the integrated densities of all the areas were obtained and compared, this resulted in data with huge variances within one pretreatment. The reason for this is most likely the inherent biological differences between patients and the brain regions used. To avoid this problem, relative densities were used to compare different brain regions and different patients.

In the literature, it has been suggested to replace hydrated autoclaving by microwave treatment. This would shorten the procedure and make it less labor-intensive. Another advantage would be that a microwave oven is cheaper than an autoclave for laboratories starting prion IHC. However, our results clearly show that hydrated autoclaving cannot be replaced by microwave treatment. In some patients, only faint synaptic staining is present (not revealed by MT), and this could potentially lead to misdiagnosis.


  Acknowledgments

Supported by the Flemish Institute for the enhancement of scientific–technological research in the industry and by the Fund for Scientific Research.

We would like to thank Dr K. O'Rourke for the kind gift of the F89/160.1.5 monoclonal antibody. We would also like to thank U. Lübke, I. Bats, S. Kumar–Singh, and E. De Leenheir for technical and photographic assistance.

Received for publication January 2, 1999; accepted June 8, 1999.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Bell JE, Gentleman SM, Ironside JW, McCardle L, Lantos PL, Doeg L, Lowe J, Fergusson J, Luthert P, McQuaid S, Allen VI (1997) Prion protein immunocytochemistry, UK five center concensus report. Neuropathol Appl Neurobiol 23:26-35[Medline]

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