The Quantitative Histochemistry of Brain

Oh, Lowry; Nr, Roberts; Ky, Leiner; Wu, Mei‐Hwan; Al, Farr; Rw, Albers

doi:10.1016/s0021-9258(18)71243-x

Cited by 186 publications

(2 citation statements)

References 8 publications

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“…The pellets were solubilized in SDS. Brain tissue contains about 100 mg protein per gram wet weight (Lowry et al 1954). When homogenizing brain tissue in water (S. Holmseth and N.C. Danbolt, unpublished), about half of the proteins will remain in the supernatant, which is virtually devoid of integral membrane proteins.…”

Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

Specificity Controls for Immunocytochemistry

Holmseth

Zhou

Follin-Arbelet

et al. 2012

J Histochem Cytochem.

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SummaryThe biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivityspecificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that crossreacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing. (J Histochem Cytochem 60:174-187, 2012)

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Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

Specificity Controls for Immunocytochemistry

Holmseth

Zhou

Follin-Arbelet

et al. 2012

J Histochem Cytochem.

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“…CONNECTION with a quantitative histochemical study of brain (6) a method was needed for measuring as little as 0.1 of cholesterol, or 1000 times less than the amount required in the Sperry method (9), for example. The reliability of the colorimetric Liebermann-Burchard reaction prompted an investigation of its fluorescence possibilities.…”

Section: Inmentioning

confidence: 99%

Fluorometric Determination of 0.1 to 10 Micrograms of Cholesterol

Albers¹,

Lowry²

1955

Anal. Chem.

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VOLUME 2 7, NO. 11, NOVEMBER 1955 1829 the end point. The addition of standard perchloric acid to the solvent corrects the small negative blank. REAGENTSPerchloric acid, 0.05 '. Dissolve ca. 4.2 ml. of 72% perchloric acid in 1 liter of dioxane and standardize against potassium acid phthalate according to the method of Seaman and Allen (7).Mercuric acetate solution. Dissolve ca. 6 grams of mercuric acetate in 100 ml. of hot glacial acetic acid (5).Nitromethane, c.p. Fisher Scientific Co. Formic acid, 98%, Fisher Scientific Co. 1,3-Di-o-tolylguanidine, O.LY. Dissolve 24 grams of reagent in 100 ml. of methanol and 900 ml. of methylchloroform. Standardize potentiometrically against 20-ml. portions of standard O.LY aqueous hydrochloric acid or 40 ml. of the 0.05.V perchloric acid in dioxane. Methanol is used as sample solvent.Dimethylformamide, E. I. du Pont de Nemours & Co.PROCEDURE Sample weights are based on an expected base content of the polymer of 5%.Determination of Total Base. Accurately weigh 0.3 to 0.5 gram of the polymer into a tared 150-ml. beaker and stir with 20 ml. of nitromethane until the material is well dispersed. Add 2 ml. of 98% formic acid, and heat until the suspension dissolves completely. Dilute with 50 ml. of cold nitromethane and allow the solution to come to room temperature. If the amine or quaternary salts present contain halide ion, add 2 ml. of 6% mercuric acetate solution.Introduce the glass and calomel electrodes into solution, set the pH meter to + mv., and titrate the solution potentiometrically with standard 0.05.Y perchloric acid. A microburet should be used.The end point of the titration is the maximum value of {AE/AV). Appropriate plots may be made.A blank must also be titrated and the titer of the sample corrected for the solvent titration.Determination of Heterocyclic Amine Salt. Dissolve a weighed 0.5-gram sample of polymer in 70 ml. of dimethylformamide. Using a glass and calomel electrode combination titrate the solution potentiometrically with the standardized solution of 1,3-di-o-tolylguanidine. The end point is determined by the maximum value of {AE/aV).The negative solvent blank is determined by titrating 70 ml.of dimethylformamide with standard perchloric acid and adding this correction to the sample titer.

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Glia, Lipogenesis, and Formation of Myelin

Korey¹

1960

Archives of Neurology

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The Quantitative Histochemistry of Brain

Cited by 186 publications

References 8 publications

Specificity Controls for Immunocytochemistry

Specificity Controls for Immunocytochemistry

Fluorometric Determination of 0.1 to 10 Micrograms of Cholesterol

Glia, Lipogenesis, and Formation of Myelin

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