Techniques to Investigate Central Nervous System Sections in Multiple Sclerosis



Fig. 1
Immunohistochemistry, confocal fluorescence microscopy, and in situ hybridization techniques. (a, b, c, bar: 1 cm) Hemispheric sections from the brain of an RRMS patient. (a) Luxol Fast Blue/periodic acid Schiff staining shows a demyelinating lesion in the white matter. (b) Staining for myelin oligodendrocyte glycoprotein (MOG) showing myelin loss in the same lesion. (c) Staining for CD68 shows strong upregulation in macrophages/microglia (inset) in the demyelinating area but also upregulation in the surrounding normal appearing white matter. (d, bar: 100 μm) Single immunohistochemical staining for CD3 (dilution 1:25) shows T lymphocytes in an acute MS lesion. Staining was performed with biotinylated anti-rabbit as secondary antibody, avidin peroxidase as third step, and DAB as substrate. (e, bar 100 μm) Same lesion as shown in (d). In this case CD3 was diluted 1:1,000, and the staining was enhanced with biotinylated tyramide. The CSA leads to immense signal enhancement with 40-fold reduction of the primary antibody. (f, bar 50 μm) Confocal fluorescence microscopy according to protocol in Section 3.3, step 3a. Staining of a demyelinating lesion in acute MS. Here, after HIER with EDTA pH 9.0, three primary antibodies derived from three species (mouse anti-transglutaminase 2, rabbit anti-Iba-1, and goat anti-GFAP) were incubated simultaneously. As a second step, we used biotinylated donkey anti-mouse, Cy3-conjugated donkey anti-rabbit, and Cy5-conjugated donkey anti-goat. The staining was finished with Cy2-conjugated streptavidin. The image shows Iba-1+ macrophages (red) and GFAP + astrocytes (blue). Transglutaminase 2 (green) is present in endothelial cells (arrowhead) and in various intensities in both Iba-1+ as well as Iba-1-macrophages but is completely absent in astrocytes. (g, bar: 20 μm) Triple staining on Alzheimer’s disease brain, performed according to protocol in Section 3.3, step 3b. First, 1 h HIER was performed with a citrate buffer (pH 6.0). After this the primary antibody anti-Aβ antibody (Ab) (1:2,000 MAB1561, clone 4G8, Millipore, Billerica, MA, USA) was incubated ON. This was followed by biotinylated sheep anti-mouse (1:500 Jackson ImmunoResearch, West Grove PA, USA) and by avidin–peroxidase (Sigma, Germany). This staining was developed with DAB substrate. The DAB precipitate, due to steric hindrance, blocks binding of further antibodies to the anti-PCNA or the biotinylated secondary antibody. After washing, sections were incubated with anti-proliferating cell nuclear antibody (PCNA) Ab (1:25,000 DAKO M0879, Glostrup, Denmark) overnight, followed by donkey anti-mouse alkaline phosphatase-conjugated Ab, and NBT/BCIP substrate to produce a blue/purple stain (arrows). In order to inactivate the binding properties of the first round of antibodies and to retrieve additional GFAP epitopes, sections were then treated with EDTA (10 mM, pH 9.0) in Tris buffer for 30 min followed by incubation with rabbit polyclonal anti-GFAP (1:1,500, DAKO Z0334, Glostrup, Denmark). Subsequently, sections were incubated with alkaline phosphatase-conjugated donkey anti-rabbit (Jackson ImmunoResearch, USA) and developed with Fast Red substrate to stain astrocytes. (h, bar: 20 μm) ISH for proteolipid protein (PLP) in combination with IHC for the protein PLP. Shown is the edge of a lesion in a case of Balo’s concentric sclerosis. At the left side, black PLP mRNA+ oligodendrocytes are seen in between red PLP+ IHC-stained myelin sheaths. At the right side, oligodendrocytes and myelin are lost. The arrowheads point at PLP+ degradation products in macrophages in the demyelinated area. ISH was developed with NBT/BCIP, while the PLP ISH was developed with Fast Red. Hematoxylin was used as a nuclear counterstain






































































































Station

Temp. (°C)

Solution

Normal-sized (2.0 × 3.0 × 0.5 cm) blocks

Double hemispheric blocks (1 cm thick) (h)

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40

50 % ethanol

30 min

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70 % ethanol

1 h

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1 h 30 min

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1 h 30 min

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1 h 30 min

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96 % ethanol

1 h

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96 % ethanol

2 h

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96 % ethanol

2 h

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Xylene

1 h

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Xylene

1 h 30 min

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3.2 Immunohistochemistry Preprocessing


For routine immunohistochemical stainings, paraffin sections are deparaffinized and dehydrated. In between these steps, we clear endogenous peroxidase. The protocol is as follows:

1.

2× 20 min deparaffinization in xylene.

 

2.

2× 96 % ethanol.

 

3.

20 min incubation in 0.2 % H2O2 in methanol.

 

4.

1× 96 % ethanol.

 

5.

1× ethanol 70 %.

 

6.

1× ethanol 50 %.

 

7.

AD.

 

Antigen Retrieval

For most antibodies, antigen retrieval is obligatory to make antigens available for recognition. Most antibodies work after heat-induced epitope retrieval (HIER), while a smaller amount of antigens is retrieved after enzymatic digestion with enzymes such as proteinase, proteinase K, or trypsin. In addition, very few antibodies may benefit from antigen retrieval with formic acid.

1.

HIER, rather than in a microwave, in our laboratory is performed in a household food steamer device (MultiGourmet FS 20, Braun, Kronberg/Taunus, Germany) by a 60 min incubation in a plastic coplin jar filled with the various retrieval buffers. Depending on the antibody, the used buffers are a citrate or EDTA–Tris buffer (pH 9. 0) as shown in Section 2. After steaming the slides are cooled down and transferred to TBS.

 

2.

Proteinase treatment is performed by incubation of deparaffinized sections with proteinase (see Section 2) for 15 min at 37 °C.

 

3.

Formic acid treatment is performed by incubation of sections for 15 min at RT.

 


3.3 Immunohistochemical Stainings




1.

Immunohistochemical single stainings.

By using antibodies against different cell types and structures, one can characterize the different lesions. The nuclear counterstain and weak background staining of the other structures in the tissue carry addition information such as the density of inflammation and the presence of edema. This information is mostly absent in fluorescence stainings which is why conventional (light microscopical) stainings are the first choice to start with. For stainings with single antibodies, we use a three-step method with peroxidase and DAB or AEC as chromogen or with alkaline phosphatase and Fast Blue or Fast Red as chromogen. After deparaffinization, blocking of endogenous peroxidase, and antigen retrieval, sections can be used for immunohistochemical stainings. Deparaffinized, untreated, or pretreated sections are transferred to TBS followed by the next protocol:

(a)

10 min preincubation with DAKO/FCS.

 

(b)

Incubation with primary antibody in DAKO/FCS overnight (ON) at 4 °C.

 

(c)

Wash TBS 3×.

 

(d)

Incubation with biotin-conjugated secondary antibody (Jackson) for 1 h at RT.

 

(e)

Wash TBS 3×.

 

(f)

Incubation with avidin–peroxidase (Sigma, diluted 1:100) for 1 h at RT or avidin–alkaline phosphatase (Sigma, diluted 1:100).

 

(g)

Wash TBS 3×.

 

(h)

Development with DAB or alternatively Fast Red or Fast Blue substrate.

 

(i)

Wash with AD 3×.

 

 

2.

Tyramide signal amplification (TSA).

Many firms sell TSA kits which are used to enhance the detection of antigens. We use TSA with a variety of antibodies. In our hands, by using TSA, the dilution of the primary antibodies can be increased 5- to 20-fold with improved staining intensity (Fig. 1d, e). The TSA protocol and the production of biotinylated tyramide (7) originally are described by King et al. (6). Alternatively, tyramide can be coupled to a range of fluorochromes as shown by Hopman et al. (8). The synthesis of biotinylated tyramide is a simple and cost-effective alternative to the purchase of TSA kits. For production of biotinylated tyramide, see Section 2.

Usage of Biotinylated Tyramide

(a)

After step f of the IHC protocol (incubation with avidin peroxidase), wash with TBS.

 

(b)

Dilute 5 μl of the biotinylated tyramide solution in 50 ml PBS and add 5 μl H2O2 (30 %); incubate sections for 10–20 min (see Note 1 ).

 

(c)

Wash with TBS.

 

(d)

Incubate sections with avidin–peroxidase (1:100) or alternatively with avidin–alkaline phosphatase (1:100) in DAKO/FCS for 30 min at RT.

 

(e)

Wash with TBS.

 

(f)

Develop with DAB, AEC, Fast Red, or Fast Blue substrate.

 

 

3.

Immunohistochemical multiple labeling protocols.

(a)

Multiple labeling with primary antibodies from different species.

Double-labeling studies mostly are performed with fluorescent dyes. However, double-labeling studies by light microscopy have certain advantages. First, it is easier to screen large areas and to trace specific areas. Second, it is easier to perform quantitative measurements. The most basic double labeling is performed with primary antibodies from different species and combination of a peroxidase and alkaline phosphatase detection system. These preferably are developed with aminoethyl carbazole (AEC) and Fast Blue as substrates. As a rule, the antibody, which in single stainings shows the weakest performance, is developed with a three-step biotin system and peroxidase as shown for single stainings. The “stronger” antibody is detected with an alkaline phosphatase-conjugated secondary antibody. In our protocol, as shown underneath, instead of peroxidase-conjugated avidin or alkaline phosphatase-conjugated secondary antibodies, also fluorescence-conjugated secondary antibodies or avidin-conjugated fluorescent dyes can be used (Fig. 1f). Since with fluorescence microscopy the number of labels are less restricted than for light microscopy (only peroxidase and alkaline phosphatase labeling, Fig. 1g), with fluorescence labeling detection of different antigens can be extended to three (Fig. 1f) or (theoretically) even more colors. Practically, however, the staining is restricted due to the limited availability of primary antibodies from various species and by the number of fluorophores which can be used without overlapping wavelengths, in most cases excitation at around 405 nm (UV), 488 nm (green “FITC-like” dyes), 543 nm (red “rhodamine-like” dyes), and 633 nm (far red dyes). Underneath is the protocol which we use for both enzymatic as well as fluorescence labeling. Primary antibodies and secondary antibodies in this protocol are incubated pairwise as follows:

Jul 12, 2017 | Posted by in NEUROLOGY | Comments Off on Techniques to Investigate Central Nervous System Sections in Multiple Sclerosis
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