Due to the requirement of huge amount of starting material, it has been difficult for establishing a ChIP assay for small tissues to detect an association of a protein with transcription factors in vivo. I have recently developed a ChIP assay for transcription factors (Sry and Sox9) that utilizes chromatin from 20 intact embryonic testes (16-18 ts stage) without cross-linking protein to DNA. All ChIP assays published so far utilize cross-linking procedure to retain protein-DNA complex throughout the procedure. Question is “Is it possible to hold your protein-DNA complex for days without perturbing its binding?”.  I say yes. My experimental conditions retained that complex. Let’s see if this ChIP assay for transcription factors (Sry and Sox9) can be validated and standardized for several other transcription factors. At the end, there will be a way out for catching up mechanisms underlying early embryonic development.

Whole-Mount Indirect Fluorescent Immunohistochemistry
Fix samples in 4% PFA (paraformaldehyde) in PBS overnight at 4°C.
Quench in 50 mM NH4Cl for 15 min.
Block for at least 24 h in blocking buffer ( 1% BSA, 0.01% TritonX, 0.02% sodium azide in PBS).
Incubate for at least 24 hour in primary antibody diluted in fresh blocking buffer.
Wash 5 times for 1h each in blocking buffer.
Incubate in secondary antibody diluted in fresh blocking buffer.
Wash 5 times for 1h each in blocking buffer.
Mount the samples.
Confocal microscopy.

Adopted from:Albrecht and Eicher, 2001 Developmental Biology 240: 92-107.

A group of Scientists (Dr. Laura O`neill and Dr. Bryan Turner of the University of Birmingham Medical School) have developed ChIP assay for small quantity of tissue samples. They used Drosophila SL2 cells as a source of carrier chromatin. One of the keys to the success of this protocol was the use of 'native' ChIP instead of the more widespread XChIP, in which proteins are chemically cross-linked to DNA before the immunoprecipitation. Chemical cross-linking is necessary to look at nonhistone proteins, which otherwise detach from the DNA during the procedure, but it tends to affect the recognition of epitopes on the flexible histone tails, thus decreasing the efficiency of immunoprecipitation of histones.

Abstract
Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. O'Neill LP, VerMilyea MD, Turner BM.  Nat Genet. 2006 Jul;38(7):835-41

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >10⁷). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as Oct4) and Cdx2) by CChIP in mouse embryonic stem (ES) cells and in inner cell mass (ICM) and trophectoderm of cultured blastocysts. Activating and silencing modifications (H4 acetylation and H3K9 methylation) mark active and silent promoters as predicted, and we find close correlation between values derived from CChIP (1,000 ES cells) and conventional ChIP (5 10⁷ ES cells). Studies on genes silenced in both ICM and ES cells (Cdx2, Cfc1, Hhex and Nkx2-2, also known as Nkx) show that the intensity of silencing marks is relatively diminished in ES cells, indicating a possible relaxation of some components of silencing on adaptation to culture.

Journal Link: http://www.nature.com/ng/journal/v38/n7/full/ng1820.html
Protocol link: http://www.epigenetics-noe.net/researchtools/protocol.php?protid=26