2. Aim of the study 17
3.4. Histological staining
References and staining methods described in detail were published by (Romais, 1989).
3.4.1. Deparaffinization
For histological staining, the paraffin from tissue sections has to be dissolved in xylene. There-fore, slides were placed consecutively in three containers of xylene for 5 min each. Afterwards, the tissue was transferred into m-xylene for 1 min. The alcohol series was performed for tissue rehydration.
3.4.2. Alcohol series
For many staining procedures, a specific hydrophilic or hydrophobic environment was required.
For tissue dehydration, slides were consecutively transferred from a more hydrophobic to a more hydrophilic solution, i.e., 2 x 100 % isopropyl alcohol, 4 min each, 90 %, 70 %, 50 % isopropyl
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3. Materials and Methods
alcohol, 3 min each and rinsed in distilled water (dH2O). For tissue dehydration the procedure was performedvice versa.
3.4.3. Haematoxylin-Eosin (H&E) staining
H&E staining was performed to obtain a general morphological overview of the tissue. Sections were deparaffinized (see section 3.4.1) and hydrated stepwise by alcohol series todH2O as de-scribed in section 3.4.2, washed in dH2O and incubated in Mayer’s hemalun solution (Merck) for 5 min. Afterwards, sections were washed withdH2O and differentiated by short dip into 1 % HCl-alcohol (1 % HCl in 90 % isopropyl alcohol). Tissue was blued by rinsing slides under tap water for 10 min. The tissue was then incubated in 1 % eosin solution (1 % eosin G, Merck, in 70 % isopropyl alcohol, stirred and filtered, 10 drops glacial acetic acid was added before use) for 5 min. Afterwards, slides were rinsed in dH2O and fast dehydrated by alcohol series until 100 % alcohol to avoid excessive elution of eosin. Next, slides were incubated for 2 x 3 min in 100 % isopropyl alcohol (two containers), 3 min m-xylene and 3 x 3 min xylene (three containers).
Slides were mounted using DePex mounting medium (Serva).
3.4.4. Luxol Fast Blue/ Periodic Acid Schiff (LFB/PAS) staining
LFB/PAS staining is used to detect demyelinized areas in EAE animals. Paraffin embedded tissue sections were deparaffinized until 90 % of alcohol (section 3.4.1 and 3.4.2). Afterwards, sections were incubated in LFB solution (0.1 % w/v LFB in 96 % ethanol, 0.05 % acetic acid) overnight at 60◦C. Sections were washed shortly in 90 % ethanol, and afterwards dipped in 0.05 % lithium carbonate (diluted indH2O,Roth), 70 % alcohol for differentiation and rinsed in dH2O. For the PAS staining, sections were incubated for 5 min in 1 % periodic acid (indH2O, Merck), then rinsed for 5 min under tap water and shortly washed indH2O. Tissue was incubated for 20 min in Schiff reagent (Sigma), rinsed under tap water for 5 min, hemalun solution (Merck) for 2 min and rinsed again underdH2O. For differentiation, sections were incubated in 1 % HCl alcohol (see above) and blued under tap water. The sections were dehydrated in increasing alcohol series and incubated in xylene (see section 3.4.1 and 3.4.2) and embedded in synthetic mounting medium (DePeX,Serva).
3.4.5. Bielschowsky silver staining
Bielschowsky silver staining was performed to determine axonal density. Sections were deparaf-finized as described earlier (section 3.4.1 and 3.4.2) and washed withdH2O. Next, sections were incubated in 20 % silver nitrate solution (inddH2O,Roth) for 20 min and stored indH2O. Mean-while 32 % ammonium hydroxide (Merck) was added dropwise to the already used 20 % silver nitrate solution while stirring, until the formed precipitate disappeared. A further two drops were then added. In this solution, slides were incubated in the dark for 15 min. Subsequently,
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3. Materials and Methods
slides were stored inddH2O containing a few drops of ammonium hydroxide. Ten drops of de-veloper (6 % formalin indH2O, 0.4 % citric acid, 0.05 % nitric acid) were stirred into the already used silver nitrate/ammonium hydroxide solution. By adding this solution quickly to the slides, a fast developing process took place. The reaction was stopped by dH2O. Afterwards, slides were incubated in 2 % sodium thiosulfate (diluted indH2O) for 2 min and rinsed shortly under tap water. The sections were dehydrated in alcohol series until xylene (see section 3.4.1 and 3.4.2) and then embedded in synthetic mounting medium (DePeX,Serva). In the stained tissue, nuclei become brown and axons black.
3.4.6. IBA1 staining
Macrophages and microglia were detected in EAE tissue by staining cell against microglia/
macrophage-specific calcium-binding protein, the ionized calcium binding adaptor molecule 1 (IBA1). For this, tissue sections were deparaffinized according to section 3.4.1 and completely rehydrated (see section 3.4.2). Slides were pretreated with citrate buffer (10 mM citric acid, adjusted to pH 6.0 with NaOH, bothMerck) 6 x for 3 min. Afterwards, the sections were cooled to room temperature (RT) and transferred into PBS. Endogous peroxidase was blocked with 3 % H2O2 in PBS for 20 min. Slides were rinsed in PBS and blocked in normal serum solution (5% serum, 5% skimmed milk powder) for one hour atRT. Normal serum origin from the same animal species as the origin of the secondary antibody,i.e., donkey serum. Afterwards, sections were incubated in primary antibody solution (1:400α-IBA1 rabbit antibody (Amersham) diluted in 50 % blocking solution in PBS) overnight at 4◦C. Sections were washed 3 x 5 min with PBS and then incubated in secondary antibody solution (1:100α-rabbit donkey antibody (Amersham) in 50 % blocking solution in PBS) for one hour atRT. Sections were again washed in PBS for 3 x 5 min and incubated in peroxidase-complex solution (1:1000 in 50 % blocking solution in PBS, Sigma) atRT for one hour. Sections were washed yet again 3 x with PBS. Afterwards, staining was developed with diaminobenzidin (DAB,Sigma) within 10 min and rinsed indH2O.
Tissues were incubated for 3 min with Mayer’s hemalum solution (Merck) for counterstaining.
Bluing of nuclei was performed by rinsing sections in tap water for 10 min. Finally, sections were rinsed indH2O, dehydrated until xylene (see section 3.4.2) and embedded as H&E stained tissue (see section 3.4.3).
3.4.7. CD3 staining
Staining of CD3, a T cell co-receptor, allows T cells to be detected in the EAE tissue. This staining procedure equals the IBA1 staining (see section 3.4.6). Here, goat-normal serum, 1:400 α-CD3 rat primary antibody (Amersham) and 1:100 goatα-rat secondary antibody (Amersham) were used.
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3. Materials and Methods
Table 3.2.:Overview of seeded cells depending on analysis
Purpose Culture dish Cell amount/well Volume of culture medium
ELISA 96 well plate 1.5 x 104 100µl
Cellular staining 4 well plate containing 5 x 104 500µl PLL coated glas slide
LumoxR slides 4 wells PLL coated 5 x 104 500µl
FACS 12 well plate 2 x 105 1 ml
Cell lysates 3 ø cm Petri dish 1 x 106 2 ml
6 ø cm Petri dish 3 x 106 4 ml