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How to Prepare a Sample.

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Mike

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Today's lesson: How to prepare a brain sample for IHC Testing of BSE.
There is "NO CHARGE" for these lessons. Donations accepted.
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Disease Disease-specific PrP – Iimmunohistochemical Immunocytochemical labelling

• Reagents

Store commercial preparations according to the manufacturer’s instructions.

Tris buffered saline Tween 20 (0.5%), pH 7.6 (TBST)

Formic acid (96%)

Citrate buffer solution (0.2%), pH 6.4

Hydrogen peroxide (30% w/v) (Sigma)

Methanol

Vector Labs, Vectastain Elite RAT IgG avidin–biotin complex (ABC) kit

Copper sulphate solution (0.5%)

Di-amino-benzidine (DAB) (Sigma)

Mayer’s haematoxylin

0.5% conc. HCl in absolute ethanol

Absolute ethanol

Xylene

Ammonia water

Primary antibody (rat anti-bovine PrP monoclonal R145 antibody, available from Veterinary Laboratoryies Agency, Weybridge, UK)

• Tissues

Formalin Formalin-fixed tissues of maximum thickness of 3 mm. Immerse in 96% formic acid for 1 hour. Rinse in running water for 10 minutes. Immerse in neutral buffered formalin for 1 hour and routinely process to wax.

• Method

Following preparation of paraffin wax embedded blocks (according to routine methods), cut sections at 3 µm thickness and mount on polysine polysine-coated slides or equivalent. Air dry and place in 60°C oven overnight.

i) Deparaffinise sections in xylene for 10 minutes.

ii) Wash in absolute ethanol twice for 30 seconds (agitate).

iii) Wash in running tap water for 10 minutes.

iv) Immerse in 96% formic acid for 5 minutes. (Formic acid treatment is used for preliminary antigen retrieval, which also maintains good tissue morphology during subsequent autoclaving. It also increases operator safety by reducing infectivity levels in the tissue being handled.)

v) Wash in running tap water for 10 minutes.

vi) Immerse in citrate buffer solution in an open plastic trough and autoclave for 5 minutes at 121°C.

vii) Cool at room temperature for 10 minutes then rinse in running tap water for 10 minutes.

viii) Immerse in 3% hydrogen peroxide in methanol for 20 minutes.

ix) Rinse in running water for 10 minutes.

x) Dry sections and isolate with wax pen.

xi) Sections should not be allowed to dry from now on.

xii) Rinse sections in TBST twice.

xiii) Apply normal rabbit serum (NRS)* for 60 minutes at room temperature. (*Dilute NRS according to manufacturer’s instructions.)

xiv) Drain off NRS and apply primary antibody (rat anti-bovine PrP monoclonal antibody R145 at 1/3000) for 16–18 hours at room temperature.

xv) Rinse sections in TBST twice for 2–3 minutes each time.

xvi) Apply secondary antibody* (rabbit anti-rat IgG) for 60 minutes at room temperature. (*Dilute secondary antibody according to manufacturer’s instructions.)

xvii) Prepare avidin–biotin complex (ABC) reagent according to manufacturer’s instructions and leave at room temperature for 30 minutes be before use.

xviii) Rinse sections in TBST twice for 3 minutes each time.

xix) Apply ABC for 30 minutes at room temperature.

xx) Rinse sections in TBST twice for 3 minutes each time.

xxi) Apply DAB – for 10 minutes.

xxii) Rinse in tap water twice for 2 minutes each time.

xxiii) Immerse in 0.5% aqueous copper sulphate for 5 minutes.

xxiv) Rinse in tap water for 1 minute.

xxv) Immerse sections in Mayers haematoxylin for 5 minutes.

xxvi) Rinse in tap water until water runs clear.

xxvii) Differentiate in 1% acid alcohol (1% HCl in ethanol) for 2–3 seconds.

xxviii) Rinse sections in tap water for 1 minute.

xxix) Immerse in alkaline ammonia water solution to convert brown haematoxylin counterstaining to blue. The same effect can be obtained by washing in running water for 5 minutes or by immersion in 0.05% lithium carbonate.

xxx) Immerse sections in absolute ethanol twice for 30 seconds each time.

xxxi) Clear sections in xylene for 2–5 minutes.

xxxii) Apply cover-slips and allow to dry.

Abnormal accumulations of PrP as shown by IHC are considered to have potential for the preclinical diagnosis of scrapie in sheep (in some, but not all, genotypes) using tonsillar (72) or nictitating membrane (64) lymphoid tissue biopsies. However, BSE infectivity has not been detected by mouse bioassay or IHC in lymphoid tissues at any time during the incubation period or clinical disease course (90), other than in distal ileum containing Peyer’s patches in experimentally infected cattle. Currently, this suggests that these tissues are unlikely to be of use diagnostically. One unpublished result of the detection of infectivity in the tonsil of an experimentally infected animal, 10 months after oral exposure, remains incomplete. The finding is in contrast to negative results in tonsils collected at earlier and later time points in the study, including clinically affected animals, and does not indicate a breakthrough in the scope for in-vivo testing of cattle.

Tomorrow's lesson: How to remove the brain sample from animal.
 

Mike

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Neil Waugh said:
Sounds good. But where the heck do you get a wax pen these days?

The new "Touchamatic Digital Wax Pen" is available at a BSE test supply center near you for the small price of $249.95 U.S. :???: :roll:
 

Neil Waugh

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You don't say. (Rips stale month of calendar. Licks pencil stub. Cypher, cypher, cypher). Yikes that 5,642.47 in Canucklehead bucks!!!!!
Might have to wait for an auction.
 

Mike

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Forget this lesson boys and girls. You can get your money back at the front desk. :roll:
 

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