Histochemical stainings were performed to visualize cells of the spinal cord (Nissl stainings) as well as muscle fibers (Hematoxylin and eosin stainings, H&E). While histochemistry describes the staining of tissues using structure specific dyes (e.g.
hematoxylin staining nuclei and eosin delineating the cytoplasm), immunohistochemical stainings are based on the use of primary- and secondary antibodies to detect a protein of interest. In a first step, the fixed section is incubated with the primary antibody. After washing steps, the fluorophore-conjugated secondary antibody is applied to indirectly detect the protein. Analysis is then performed under UV light excitation using a microscope equipped with respective filter sets. Alternatively, HRP-conjugated secondary antibodies can be used, whereby HRP converts the substrate 3,3'-Diaminobenzidine (DAB) into an insoluble brown reaction product in the presence of H2O2. The signal can then be detected using a common transmission filter.
4.12.2.1 Nissl stainings
For Nissl stainings the basophilic organic compound Cresyl violet is used to outline nucleic and ribosomal structures of nerve tissue. In this work, Nissl stainings were exclusively performed on 7 µm thick spinal cord sections. First, spinal cord sections were deparaffinized by incubation in Xylol for 3 min. Via incubation in decreasing EtOH concentrations (100 %, 96 %, 70 %, 50 %, 3 min each) and subsequent incubation for 5 min in PBS, the sections were rehydrated before they were stained in Cresyl violet acetate for 10 min. Following a short wash in H2O, sections were washed in highly diluted acetic acid (2-3 drops in 100 ml H2O), dehydrated via increasing EtOH concentrations (50 %, 70 %, 96 %, 100 %, 3 min each), air-dried and embedded in Entellan mounting media.
4.12.2.2 Hematoxylin and eosin (H&E) stainings of muscle fibers
H&E stainings were in this study performed to counter-stain mouse muscle tissue for subsequent fiber size measurements. In order to stain with H&E, muscle sections of 7 µm thickness were deparaffinized by incubation in Xylol for 30 min. Following incubation in decreasing EtOH concentrations (100 %, 96 %, 70 %, 50 %, 3 min each), the sections were quickly washed once in PBS and then incubated in H2O for 1 min. Next, sections were incubated in Hematoxylin for 6 min and afterwards washed in H2O for 15 min. The sections were rinsed quickly in H2O once to remove excess dye and then placed into Eosin solution for 1 min. To clarify the stainings, sections were rinsed in H2O 6-7 times and then dehydrated in increasing EtOH concentrations. Finally, sections were air dried and embedded in Entellan.
4.12.2.3 Immunohistological stainings of motor neurons
In order to stain motor neurons, 7 µm thick spinal cord sections were deparaffinized for 2 h in Xylol and subsequently rehydrated in decreasing EtOH concentrations (100 % I, 100 % II, 96 %, 70 %, 1 min each). Following an incubation in H2O for 10 min, antigen retrieval was performed by boiling the sections in citrate buffer for 3 x 5 min at 600 W in a microwave.
After boiling, the sections were left in citrate buffer and cooled down to RT for 45 min. To remove citrate buffer, sections were washed once in TBS for 5 min. Blocking of the sections was carried out in 5 % of the secondary antibody`s host serum plus 5 % BSA in TBS by directly pipetting the solution on the sections and incubating for 45 min at RT in a wet chamber. Subsequently, sections were washed for 3 x 5 min using TBS in a glass tray. After that, the sections were incubated with primary antibodies diluted in blocking media o.n. at 4°C in a wet chamber (Rabbit α-PLS3 1:40; rabbit α-V5 1:40, goat α-Chat 1:40). The next day, unbound primary antibody was removed from the sections by 3 x 5 min washing steps in TBS. The secondary antibodies were diluted in freshly made blocking solution (Donkey α-goat Alexa 488 1:200, donkey α-rabbit Alexa 568 1:200) and directly pipetted on the sections. Incubation with the secondary antibodies was carried out for 3-4 h at 4°C in a wet chamber. To get rid of unbound secondary antibody, sections underwent 3 more washes for 5 min each. To remove residual salt, sections were briefly rinsed in H2O and then embedded in Mowiol® mounting media.
4.12.2.4 Immunohistological stainings of neuromuscular junctions (NMJ)
Neuromuscular junction (NMJ) stainings were either performed on proximal- (Transversus abdominis, TA) or distal muscle tissue (Gastrocnemius). The isolation of TA- and Gastrocnemius muscle is described in chapter 4.14.2.2. All washing and incubation steps were performed in 24 well plates.
First, the tissue derived from TA muscle was fixed for 10 min and such from Gastrocnemius muscle for 20 min in 4 % PFA at RT. After fixing, the TA muscle could directly be used for stainings. Different from that Gastrocnemius was embedded in 4 % low melting agarose and sliced into 150 µm thick sections prior the actual staining protocol. From this point on, the staining protocol was the same for TA- and Gastrocnemius muscle. To remove excess PFA, the muscles were washed in 1 x PBS 3 x for 10 min on a rocking platform at RT. To permeabilize the tissue, muscles were incubated in PBS containing 2 % of Triton X for 30 min on a rocking platform. Following permeabilization, blocking was performed in PBS containing 4 % BSA and 1 % Triton. Finally, the antibodies were prepared in blocking solution (Mouse-α-2H3 1:100, mouse-α-SV2 1:100, mouse-α-neurofilament 1:250) and pipetted on the tissue samples. To obtain the best results, incubation with the first antibody was always performed o.n.. To reduce background, the samples were 6 x washed in 1 x PBS for 10 min under constant shaking on a rocking platform. At this point of the protocol, a Bungarotoxin (BTX, labeled with Rhodamine) staining step was included into the protocol. For that, 10 µl of BTX stock solution (1 M) was diluted in 7 ml of PBS and the samples incubated for 10 min under shaking at RT. In the meanwhile, the secondary antibody (goat α-mouse Alexa 488 1:250) was diluted in PBS. After the BTX staining had completed, the media was exchanged with the secondary antibody in PBS without any intermediate washing step and incubated for 2 h at RT on a rocking platform and wrapped in tin foil.
4.12.2.5 Immunohistological stainings of murine embryonic fibroblasts (MEF)
For staining of murine embryonic fibroblasts, cells were trypsinized and replated in 12 wells provided with sterile cover slips. After settling o.n., MEFs were rinsed in PBS once and afterwards fixed in 4 % PFA for 15 min at RT. Following fixation, the cells were washed once in 1 x PBS for 5 min. To make the epitope better accessible for the antibody, antigen retrieval was conducted by adding 80°C hot citrate buffer (pH 6.0) to the cells and incubating them under constant cooling to RT for 20 min. After another washing step in 1 x PBS, cells were permeabilized in 0.2 % Triton X (alternatively Tween20) in PBS for 5 min. For blocking, cells were incubated in 5 % of the secondary antibody`s host serum plus 5 % BSA in PBS for 2 h at RT. Next, the primary antibodies (Rabbit α-PLS3 1:40, mouse α-V5, rabbit α-V5 1:40, mouse α-Vinculin 1:150) were diluted in 5 % BSA in PBS and given to the cells for o.n.
incubation. The next day, unbound antibody was removed in 3 washing steps in 1 x PBS for 15 min each. The secondary antibodies (Donkey α-rabbit Alexa 350 1:250, goat α-mouse Alexa 488 1:250) were codiluted with Phalloidin (1:40) in PBS containing 5 % BSA. For an optimum result, the cells were incubated in secondary antibody/phalloidin for 4 h, however, if shorter incubation times were necessary, the time could be reduced to a minimum time of 1 h. To remove residual salt, cover slips were repeatedly dunked in dH2O before they were
mounted in Vectashield mounting media containing DAPI. Especially when an Alexa 350 conjugated antibody was used, the DAPI of the mounting media could mask all upcoming signals. In this case, DAPI-free Mowiol was used as mounting media.
To save antibodies, in all experiments a 40 µl droplet of the respective antibody solution was pipetted on parafilm and the cover slips placed on the drop with the cells facing the solution. For washing steps, the cover slips could easily be transferred into 12 wells again.