2 курс / Гистология / Введение_в_иммуноцитохимию_Полак_Дж_,_Ван_Норден_С_

Приложение

63

Иммуноцитохимия (см. рис. 15).

Инкубацию удобно вести

в

многоячеечных

планшетах.

В каждую ячейку

планш ета

умещается 15

мкл раствора.

Предпочтительно

профильтровать

все буферы

и

растворы,

устанавливаю т в стакане с буфером промывки

так,

чтобы

было возможно слабое перемешивание. Во

время

окраш ива­

ния сеточки

нельзя осушать.

1. Обработайте срезы 10%-ным раствором

перекиси

водоро­

да, чтобы сделать смолу проницаемой.

2.

Промойте

профильтрованной

дистиллированной водой.

3.

Удалите

избыток жидкости

с сеточки и

поместите ее на

местите ее в раствор

первых

антител,

приготовленных

на

ЗФ Ф Р

с 0,1%

БСА

и 0,1% азида натрия,

на 1 ч, если инку­

бация

ведется

при

комнатной

температуре, или на

48 ч

при

4°С. Оптимальное разведение

антител определяют

экспери­

ментально-.

5. Тщательно

промойте

0,05 М трисовым

буфером,

pH

7,2.

содержащ его 1 % БСА на пять минут.

8. Приготовьте раствор вторых антител, меченных

золотом

(например, козьих антител к IgG

кролика) в 0,05

М трисо-

вом буфере, pH 8,2, содержащем

1 % БСА, и путем центри­

фугирования в течение 20 мин при

2000 g удалите микроагре­

буфера, pH 7,2, содержащ его

0,2% БСА, затем буфером

без^

БСА и дистиллированной водой.

11. Осушите сеточки бумагой,

адсорбирующей жидкость.

Д о ­

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