Immunofluorescence Analysis of Cellular Proteins

Semithin (about 0.1 μm) ultramicrotomic sections were obtained from the resin blocks and used for immunofluorescence confocal and phase contrast microscopy. After washing with PBS, sections were incubated overnight with primary and then with secondary antibodies as described [23 (link)]. Primary antibodies against 19S, 20S, and polyubiquitinated proteins are the same used for electron microscopy. Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse or anti-rabbit IgG (Life technologies, Paisley, UK). Specimens were analyzed by a TCS SP5II confocal laser scanning microscope equipped with PL APO 40x/1.25 NA and 63/1.40 NA oil-immersion objectives (Leica, Heidelberg, Germany).

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Pecci A., Necchi V., Barozzi S., Vitali A., Boveri E., Elena C., Bernasconi P., Noris P, & Solcia E. (2015). Particulate cytoplasmic structures with high concentration of ubiquitin-proteasome accumulate in myeloid neoplasms. Journal of Hematology & Oncology, 8, 71.

Publication 2015

Alexa Fluor 488-conjugated anti-mouse or anti-rabbit IgG TCS SP5II confocal laser scanning microscope

Alexa fluor 488 Anti igg Antibodies Confocal laser scanning microscope Electron microscopy Immersion Immunofluorescence Mouse Phase contrast microscopy Proteins Rabbit Resin Ultramicrotomic

Corresponding Organization : Istituti di Ricovero e Cura a Carattere Scientifico

Other organizations : Policlinico San Matteo Fondazione

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Variable analysis

independent variables

Primary antibodies against 19S, 20S, and polyubiquitinated proteins

dependent variables

Immunofluorescence confocal microscopy
Phase contrast microscopy

control variables

Semithin (about 0.1 μm) ultramicrotomic sections obtained from the resin blocks
Incubation of sections with primary and secondary antibodies
Specimens analyzed by a TCS SP5II confocal laser scanning microscope equipped with specific objectives

positive controls

Primary antibodies against 19S, 20S, and polyubiquitinated proteins are the same used for electron microscopy

negative controls

No negative controls explicitly mentioned

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