Immunostaining materials

McDermott, N., Farah, N., and Milburn, C. 1997. MIB1 staining in archival material problems with immunostaining in older paraffin-embedded tissue may limit its predictive value. J. Cell. Pathol. 2 113-115. 

Mouledous, L., Hunt, S., Harcourt, R., Harry, J.L., Williams, K.L. and Gutstein, H.B. (2003) Proteomic analysis of immunostained, laser-capture microdissected brain samples. Electrophoresis 24,296-302. Nagy, A. and Delgado-Escueta, A.V. (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114-1123. 

Nilsson G, Wang M, Wejde J, et al. Detection of EWS/FLI-1 by immunostaining. An adjunctive tool in diagnosis of Ewing s sarcoma and primitive neuroectodermal tumour on cytologi-cal samples and paraffin-embedded archival material. Sarcoma. 1999 3(l) 25-32. 

Mercury deposits This is observed as a black precipitate which is spread randomly across the specimen. It can be corrected by removing the mercury through dezenkerization of B 5-fixed material prior to immunostaining. 

Fig. 2. Histochemical staining of control and VeMi-null aorta for elastin, mucins, and smooth muscle. (A) Control aorta stained for elastin with Hart s stain showing 5-6 elastic lamellae and (B) Ae 7-null aorta showing the abnormal separation between the elastic lamellae. (C) Control aorta stained for sialomucins with Alcian blue (blue) with elastic lamellae appearing opaque and (D) Neul-miR aorta stained for sialomucins indicating the large mass of positive material between elastic lamellae. (E) Control aorta immunostained for smooth muscle with an antibody to alpha-smooth muscle actin (red) showing the thin area between elastic lamellae and (F) Afe 7-nuU aorta showing the increased mass of smooth muscle between elastic lamellae, whereas some areas were devoid of smooth muscle (arrow). Scale bar, 22 pm. From Starcher et (See Color Plate 46.)...

Figure 4, Development of affinity-purified rabbit mti-y-H2AX antibodies. Rabbits were immunized, sera were collected, and immunostaining was performed using nonirradiated and irradiated HeLa cells as described in Materials and Methods. (A) Raw serum collected at 51 or 86 days postimmunization, as indicated. (B) Panels labeled TocV show immunostaining with antibodies obtained by y-H2AXphosphopeptide affinity chromatography of serum from 5 Id bleed of rabbit 1663. Cells were treated with 300 cGy or no radiation as indicated. Phosphopeptide competitor (1 pg/ml) was present where indicated. Panels labeled DAPI" show counterstainingfor DNA.

Protocol 5.2 describes a methanol-acetone fixation technique (Pisano et al. 1993 Cenci et al. 1994) which results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation protocol results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is a poor preservation of chromosome structure. In most instances, the chromosomes do not exhibit a distinct morphology and tend to coalesce into one or more masses of chromatin. 

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