Items Tagged ‘Fero-Histology’

August 1st, 2016

Fluorescent in Situ Hybridization

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M. Fero 4/6/2006 (courtesy of Katie Rudd, Trask lab) Materials 20 ng BAC DNA clone grown o.n. in 5 mL LB + chloramphenicol Autogen reagents Slide warmer Organics: MeOH, EtOH, formamide Fixative (3:1 MeOH:glacial HOAc) Biotin (or digoxigenin) nick-translation Kit (Invitrogen) 20x SSC (3 M NaCl, 0.3 M NaCitrate, pH 7.0) TE, pH8 CoT DNA […]

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August 1st, 2016

Anti-GFP/BrdU In Situ Immunostaining

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<R. Gu 4/6/2005 Materials 24 well dishes Millicell 0.4 µm inserts (Millipore PICM01250) – coated with fibronectin Culture medium PBS Fixative: 4% paraformaldehyde in PBS (pH 7.6) Rabbit anti-GFP (Molecular Probes, A-11122) FITC-goat anti-rabbit IgG (Jackson ImmunoResearch, 111-096-006) Biotinylated anti-BrdU (AbCam, ab2284-125) Streptavidin-Alexa Flour 555 (Invitrogen/Molecular Probes, S21381) Goat serum Horse serum BSA 2N HCl […]

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August 1st, 2016

Immunostaining with Anti-Digoxigenin Alkaline Phosphatase

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Modified from C.H. Lin 5/2005 Materials Anti-digoxigenin-AP conjugate (Roche #11093274910, 150U/200 uL, $168) Levamisole (Sigma #T1512 , 2 g, $10) (blocks endogenous phosphatase activity) BCIP (Roche #11383221001, 3 mL, $49) NBT (Roche #11383213001, 3 mL, $48) Atlternate BCIP/NBT (Roche, #11697471001, 20 Tabs, makes 10 mL/Tab, $67) Blocking Reagent (Roche #11096176001, 50 g, $104, or 10x […]

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August 1st, 2016

Immunohistochemistry (IHC) of Thymus Frozen Sections

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(courtesy of Farr lab, UWMC) Materials Cold Acetone (-20ºC) in Coplin jars PBS, BenchKote paper, plastic lids Anti-digoxigenin-HRP diluent: 1% BSA in PBS (store 10 mL aliquots of BSA at -20ºC) Anti-rat-digoxigenin diluent: 5% Milk powder, 10% mouse serum, 10% goat serum, 1% BSA in PBS Primary and secondary antibodies (this had a link that […]

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August 1st, 2016

X-gal Staining Procedure

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M. Fero — 3/24/04 — adapted from P. Soriano Materials Fixative 2% Paraformaldehyde (in PBS) Staining solution 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCI, 0.01% sodium deoxycholate 0.02% Nonidet P-40 (NP-40) in PBS. X-gal Stock 40 mg/mL X-Gal (5 -Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) in dimethylformamide store at -20°C in small aliquots. X-gal Mix (make […]

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July 15th, 2016

BrdU-Immunoperoxidase Staining of Paraffin Tissue Sections

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M. Fero Materials Syringes, 1 mL with 36G needles BrdU: 3-10 mg/mL in PBS Fixative: Methyl Carnoy’s, or 4% paraformaldehyde in PBS Paraffin processing materials Organic solvents: Xylene, EtOH, MeOH 0.3% H2O2: 2 mL 30% H2O2 in 200 mL MeOH Trypsin: Dilute (10x stock, -20ºC) 1/5 in PBS 2.5 N HCl: Dilute concentrated HCl 1/22 […]

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July 15th, 2016

Hematoxylin and Eosin (H&E) Staining

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(M. Fero, courtesy of the Porter lab) Revised 2/07 Also see Wei-Ming and Emily’s protocol  This reference link is broken. Reagents Stains (change after every 3rd use) Harris hematoxylin: 1X stock. Anatech Lmtd. Cat #842. (616) 964-6450 Gill’s Hematoxylin: See recipe. Eosin: 1x stock. Anatech Cat #837. Solutions (make fresh each day) Acid alcohol: 75% […]

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July 15th, 2016

Necropsy Form

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Necroscopy Notes Form

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July 15th, 2016

Tissue Staining

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Deparafinization The paraffin is removed from the section with xylene. If the tissues are to be stained with an aqueuous solution then the slides must rehydrated in graded ethanol baths. Unless a time is indicated the approach is to gently agitate the slides by repeated immersion ~20x in each bath: Procedure Xylene for 2 min. […]

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July 15th, 2016

Tissue Processing and Sectioning

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M. Fero 12.23.2011 Fixation The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). 10% NBF contains 4% formaldehyde by weight and is therefore equivalent to 4% paraformaldehyde. NBF contains a pH buffer plus a preservative (methanol) which prevents the conversion of formaldehyde to formic acid. Because of the preservative, NBF has a […]

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July 15th, 2016

Complete Mouse Necropsy

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Euthanasia Euthanasia and mouse necropsies require prior IACUC approval. The mode of euthanasia should be chosen which minimizes pain or distress to the animal and provides biologically meaningful samples. Only personnel with appropriate AHR training may handle live animals. Isoflourane inhalation: This should be given in sufficient dose to cause complete loss of breathing and […]

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July 15th, 2016

Immunoflourescence of Cultured Cells

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M. Fero — 3/13/98 Materials L-lysine coated glass cover slips or charged glass slides Neutral Buffered Formalin (Sigma HT50-128) 0.5% NP40 in PBS (2.5 N HCl or 0.07 N NaOH for BrDU staining only) Primary and secondary antibodies Vectashield (Vector Labs) Protocol Grow cells on L-lysine coated glass slips or cytospin cells onto charged glass […]

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