Citraconic anhydride solution

ANTIGEN RETRIEVAL PROTOCOL WITH USE OF CITRACONIC ANHYDRIDE SOLUTION... 

A boiling that heats FFPE tissue sections in 0.05% citraconic anhydride solution for 10-15 min may achieve identical IHC results as that obtained by heating at 98°C for most antibodies (90%) however, nonspecific background may increase. 

Acid Curing. Urea-formaldehyde resins and resol-phenol-formaldehyde resins can be acid-cured by wastes from the production of maleic anhydride [1902]. The waste from the production of maleic anhydride contains up to 50% maleic anhydride, in addition to phthalic anhydride, citraconic anhydride, benzoic acid, o-tolulic acid, and phthalide. The plugging solution is prepared by mixing a urea-formaldehyde resin with a phenol-formaldehyde resin, adding the waste from production of maleic anhydride, and mixing thoroughly. 

Three pH values (acidic, neutral, and basic AR solution), and three heating conditions (under boiling, boiling, and pressure heating) are recommended for the basic test battery. However, alternative procedures may be applied according to laboratory facilities and routine protocols as described above. Currently, citrate buffer pH 6.0, Tris-EDTA buffer pH 8-9, and certain AR solutions at lower pH, such as boric acid pH 2-3, or acidic acid buffer pH 2, as well as 0.05% citraconic anhydride pH 7.4, may be used to evaluate the optimal AR protocol. 

If this antibody was successfully used, to repeat the protocol of AR-IHC with certain tested tissue slides may be necessary to demonstrate the IHC result. If IHC staining result is not satisfactory, further steps are followed by testing various AR solutions with different pH. For convenience, try to use commonly used solutions such as citrate buffer of pH 6.0,Tris-HCl buffer plus EDTA at pH 8 or 9,0.05% citraconic anhydride at pH 7.4. In some cases, low pH of 1-2 (acetic buffer or other type of buffer solutions) may be tested in order to find out the optimal pH value that produces the best IHC result. 

As documented by numerous publications, the key factor of heat-induced AR is high temperature heating formalin-fixed, paraffin-embedded (FFPE) tissue sections in water solution. For some antigens, certain optimized pH value of the AR solution and/or adjusted temperature may be required to reach the best result by the use of the test battery approach mentioned above. In 2005, Namimatsu et al. reported a novel AR solution containing 0.05% citraconic anhydride, pH 7.4, for heating FFPE tissue sections at 98°C for 45 min to... 

Citraconic anhydride also has been used to reverse the effects of formalin fixation in tissue sections. Namimatsu et al. (2005) found that heating deparafinized tissue sections in a dilute solution of citraconic anhydride broke the formaldehyde crosslinks and restored antigen recognition of proteins within the samples. 

Itaconic acid has been prepared by the distillation of citric acid,2 of aconitic acid,3 and of itamalic add 4 by heating dtric acid with dilute sulfuric acid in a closed tube 5 by treating aconitic acid with water at 1800 6 by heating citraconic acid with sodium hydroxide 7 by heating citraconic anhydride with water at 1500 8 and by heating a concentrated solution of citraconic add at 120-130° in a sealed tube.9... 

A mixture of 100 g. (0.88 mole) of citraconic anhydride (p. 28) (Note 1), 100 cc. of water, and 150 cc. of dilute nitric acid (1 part of concentrated nitric acid to 4 parts of water by volume) is evaporated in a 500-cc. Erlenmeyer flask until the appearance of red fumes (Note 2). The solution is cooled and the mesaconic acid is collected on a filter. The mother liquor is evaporated to 150 cc., cooled, and the crystalline solid which separates is collected on a filter. Further concentration of the mother liquor to 50 cc. yields more product (Note 3). The entire product is recrystallized from roo cc. of water. The yield of product melting at 203-205° is 50-60 g. (43-52 per cent of the theoretical amount). 

Mesaconic acid has been prepared by heating citraconic acid with dilute nitric acid, with hydriodic acid,2 or with concentrated sodium hydroxide solution 3 by heating a concentrated water solution of itaconic or citraconic acid at 180-200° 4 by treating citradibromopyrotartaric acid and mesodibromopyrotartaric acid with potassium iodide and copper at 1500 5 and by heating citraconic anhydride with nitric acid.6... 

Add a quantity of citraconic anhydride to the reaction medium to provide at least a 5—10 molar excess of reagent over the amount of amines to be modified. Even greater molar excesses may be required for total blocking of all the amines of some proteins. When adding citraconic anhydride, multiple additions may be done to maintain solubility of the reagent in the reaction solution. 

Isolation of Proteins with a Reduced Nucleic Acid Level. The procedure is virtually identical to that described for succinylation of yeast proteins (87). In a typical experiment proteins, together with NA, were extracted from the disrupted yeast cells at pH 8.5-9.0 and centrifuged at 15,000 rpm for 30 min at 5°C. Citraconic anhydride then was added in small increments to the supernatant with constant stirring while the pH was maintained between 8.0-8.5 by adding 3.5IV NaOH. After the stabilization of the pH, the pH of the solution was decreased to 4.2 to precipitate the proteins. Protein then was separated by centrifugation, dissolved in water (pH adjusted 8.5), dialyzed extensively against water (pH 8.5) at 5°C, and lyophilized. 

This bond strength is considerably higher than any value for the adhesion to smooth dentin surfaces which has been previously reported. The bond strength is slightly decreased when more dilute citric acid solutions are used. Other pretreatments yielding tensile adhesion values above 10 MPa include solutions of 1% alcoholic (1 2) citraconic anhydride, 1% alcoholic tetrahydrofuran -2,3,4,5-tetracarboxylic acid, and 1% aqueous ethylenediamine-tetraacetic acid tetrasodium salt. 

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