ID4 Antibody - Azide and BSA Free

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• ID4 Antibody

  Name: Anonymous

  Application: Immunohistochemistry-Frozen

  Sample Tested: Testis

  Species: Opossum

  Verified Customer | Posted 01/05/2017

  Tested in gray short-tailed opossum (Monodelphis domestica) in testis from 8-month animal. Tissue was fixed in 4% Paraformaldehyde overnight, incubated in 30% sucrose/PBS overnight and embedded in OCT. Sections were cut on a cryostat at 10um. For immunofluorescence staining: Slides were dried 15 minutes, washed 3x3 min TBST (0.1% Triton X-100), permeabilized for 10 minutes in 2% Triton X-100 in TBS, rinsed 3x3 min TBST, blocked in 10% heat inactivated goat serum in TBST for 30 min, and primary added at 1:100 in 10% blocking solution. Slides were coverslipped with parafilm overnight at 4C. The next day slides were washed with TBST 4x5 min, secondaries (AlexaFluor 568 goat anti rabbit and AlexaFluor 488 goat anti mouse 1:500) for 1 hour, 3x3 washes TBST, and mounted with VectaShield HardSet (+ DAPI). Intense staining at periphery of seminiferous tubules of Uchl-1 (red in image) with these conditions. 1:200 also worked though staining was less intense (not shown). The same conditions with 1% Triton X-100 for permeabiliziation resulted in weak staining at 1:100 and 1:200. However, ID-4 did not work well under any conditions. Shown in green is some faint staining in interstitial cells but this was not consistent between samples and did not appear to be specific. ID-4 was tested in 4 and 8 month opossum testes, and at dilutions ranging from 1:25 to 1:500. Single-antibody staining was also performed for each antibody and successful in Uchl-1, not ID-4.

  

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