Immunohistochemistry methods

2.15.1 Preparation of slides

Slides were coated in adhesive agents with one of three types of coating to prevent brain section loss during the immunohistochemical procedures. Poly-L- lysine and Vectabond™ pre-coated slides were purchased from Vector Labs (Peterborough, UK). Poly-L-lysine coated slides were used with all control brain and Huntington’s disease brain samples from the Cambridge Brain Bank. Vectabond™ pre-coated slides were used for R6/2 HD and control transgenic mouse brain sections. All other slides were coated in 3- aminopropyltriethoxysilane (APES, Sigma). This was done by first de-greasing slides by washing them in hot detergent (Hospec, Young’s detergent, Bolton, UK) and rinsing them in 100% EtOH and then ddHgO and air-drying for 10 mins. The slides were then immersed for 2 minutes in a freshly prepared 2% solution of APES in acetone. The slides were briefly drained and then washed in ddHgO before drying overnight at 40°C.

2.15.2 Mounting brain sections

All HD brain and matched control sections were the kind gift of the MRC Cambridge Brain Bank. Formalin fixed and paraffin embedded human control sections for optimising the procedures were kindly donated by Dr J McLaughlin from the Dept, of Histopatholgy, RFH. Transgenic mouse brains were fixed in formalin and embedded in paraffin by Mr John Difford in the Dept of Pathology, RFH. Paraffin embedded brain blocks were sectioned (10pm) using a Leica microtome, allowed to dry initially at 37°C, and then overnight at 55°C. 7pm frozen sections were cut on a CM 1900 Leica cryostat and mounted on poly-L- lysine coated slides, air-dried for 30 min at room temperature (RT), fixed in acetone for 10 min and again air-dried for 30 min. Paraffin embedded sections were stored at RT whilst frozen sections were stored at -20°C.

2.15.3 Antigen retrieval methods for paraffin embedded sections For each new antibody used with paraffin embedded section the following antigen retrieval techniques were used to assess optimum conditions. These were all performed before the serum blocking stage. All chemicals were from Sigma, UK.

0.1% trypsin in ddHgO for 12 minutes on slide at RT, then 0.1^g/ml soybean trypsin inhibitor in PBS for 5 minutes to terminate the reaction.

0.1% pepsin in 0.01 M HCI, pH2.3 for 15 minutes at RT, followed by 3 five minute washes in ddHgO .

0.02Units/ml Neuraminidase in PBS for 60 mins at RT followed by three washes in PBS.

Sections were placed in citrate buffer lOmM, pH6 and heated in a water bath at 97°C for 2 hours, then section were washed in PBS.

Sections were heated in a 2L bath containing citrate buffer lOmM in the microwave (High setting) for 20 minutes.

2.15.4 Basic Immunohistochemistry protocol

The avidin-biotin-complex technique (ABC) was used throughout with both horseradish peroxidase (MRP) and alkaline phosphatase (AP) conjugates. Peroxidase was demonstrated using 3’diaminobenzidine (DAB, Sigma) chromagen and alkaline phosphatase was demonstrated using the Vector SK5100 kit with 0.1M levamisole.

2.15.4.1 ABC horseradish peroxidase (HRP) system with diaminobenzidine hydrochloride (DAB) for paraffin embedded sections

Paraffin embedded sections were de-waxed by sequential washes (three times each) in : xylene, 100% EtOH, with a final wash in ddHgO. To remove endogenous peroxidase sections were incubated in 0.3% HgOz/methanol for 30 mins, then washed for lOmins in ddHgO. At this stage the appropriate antigen retrieval was performed (see 2.5.13) then sections were washed in PBS. Blocking was achieved by incubation with 10% v/v normal goat serum (DAKO)/PBS for BOmins at RT in a humidified chamber. Sections were then drained and the primary antibody applied and incubated in a humidified chamber with incubation time and temperature optimised for each antibody (see 5.2.2.4;5.2.3.5). The sections were washed in PBS and incubated with the appropriate biotinylated secondary antibody for BOmins at RT. This was followed by three washes in PBS and the AB Complex (DAKO) was applied to the sections for BOmins at RT. To prepare the DAB substrate (Sigma) 2.5mg of DAB was dissolved in 5ml PBS to which 50|il of 0.3%Hz02 was added. Sections were then incubated with this solution and observed for staining intensity (takes from

5-10 mins and observed by direct visualisation). The sections were then washed for lOmins in running tap HgO, then nucleii were counterstained with Meyer’s haematoxylin and differentiated in acid/alcohol/0.5% HCI in 70% EtOH (this removes the cytoplasmic colour and leaves the stained nuclei). Sections were dehydrated in 95%/100% EtOH and cleared in xylene before mounting in DPX (contains xylene, Agar Scientific Ltd. Cambridge UK).

2.15.4.2 ABC Alkaline phosphatase system for frozen sections

This was used because it produced a sharp red colour with positive antibody staining in frozen sections with little background.

Blocking was achieved by incubation with 10% v/v normal goat serum (DAKO)/PBS for 60mins at RT in a humidified chamber. Sections were then drained and the primary antibody applied and incubated in a humidified chamber with incubation time and temperature optimised for each antibody (see 5.2.2.4; 5.2.3.5). The sections were washed in PBS and incubated with the appropriate biotinylated secondary antibody for GOmins at RT. This was followed by three washes in PBS and the AB Complex (DAKO) was applied to the sections for GOmins at RT. The sections were then rinsed in PBS for 5 minutes, then the alkaline phosphatase was demonstrated using the Vector SK5100 kit (Vector Labs, according to manufacturer's instructions) with 0.1M levamisole (Sigma). Nucleii were counterstained with Meyer’s haematoxylin and differentiated in acid/alcohol/0.5% HCI in 70% EtOH before dehydration in 95%/100% EtOH and clearing in xylene before mounting in DPX.