Immersion fixation can produce useful corroborative evidence but any micrographs from either fixation must show resolvable unit membranes if it is to be used to support a mechanism for MFGM formation. BTN has previously been localized with immunocytochemistry to the apical plasmalemma of the mammary cell in several mammals and around the MFG in the alveolus (Mather and Jack 1990, 1993; Heid and Keenan 2005), but no one has been able to demonstrate any cytoplasmic label indicating the route to the plasmalemma. We now have more direct evidence for the involvement of the Golgi vesicles with BTN synthesis and transport using Light Microscope (LM) immunocytochemistry. strong class=”kwd-title” Keywords: alveolar epithelial biology, cellular localization, electron microscopy, immunocytochemistry, secretory pathway Introduction Despite numerous investigations over many years, there is still no consensus as to the mechanism of secretion of the milk fat globule (MFG) or the structure of the milk fat globule membrane (MFGM) after secretion. The individual proteins and phospholipids involved have been comprehensively investigated, and butyrophilin (BTN), xanthine oxidase (XO), adipophilin (ADP/Plin2) are generally accepted as the building LFM-A13 blocks of the MFGM; however, the ways in which they interact to form the MFGM are still controversial (McManaman et al. 2002; Heid and Keenan 2005; Chong et al. 2011; Mather 2011). We have recently demonstrated that the biochemical structure of BTN is consistent with processing via the Golgi system (Jeong et al. 2013). This would support the electron microscopical observations of Wooding (1971, 1977) that, in all species examined, a significant area of Golgi secretory vesicle membrane associates with the perimeter of the intracellular apical lipid droplet to preform patches of a structure identical to the MFGM around the newly secreted, alveolar MFG. This general observation was reinforced by the finding that, under certain conditions in the goat mammary gland, the MFGM could form intracellularly by the fusion of lipid-associated Golgi vesicles with each other without the participation of the apical plasmalemma (Wooding 1975). This produces a large intracellular vacuole containing a MFG bounded by a MFGM. The conventional fusion of the Golgi vesicles with the plasma membrane would allow for the patches of membrane associated with the lipid to aggregate into the continuous unit membrane separated from the lipid by a uniform, 15-nm of material that is continuous LFM-A13 with the LFM-A13 cytoplasm. If two Golgi vesicles separated by a cytoplasmic bleb fuse simultaneously with the plasma membrane, this would produce the signet ring MFGM, which can be found in a Tmem14a small variable percentage in all milks so far investigated (Wooding 1977). This scenario takes into account all of the EM observations in a variety of speciescow, ewe, goat and rodentsas long as the material is fixed by aldehyde perfusion as soon as practical after the death of the animal. Any fixation can produce artefacts; but if the perfusion produces uniform fixation with resolvable membranes, the results are always more reliable than those achieved with immersion fixation. Immersion fixation must produce a variety of fixation quality from the edge to the middle of the block, and this seems especially true for lipid-laden tissue. Immersion fixation can produce useful corroborative evidence but any micrographs from either fixation must show resolvable unit membranes if it is to be LFM-A13 used to support a mechanism for MFGM formation. BTN has previously been localized with immunocytochemistry to the apical plasmalemma of the mammary cell in several mammals and around the MFG in the alveolus (Mather and Jack 1990, 1993; Heid and Keenan 2005), but no one has been able to demonstrate any LFM-A13 cytoplasmic label indicating the route to the plasmalemma. We now have more direct evidence for the involvement of the Golgi vesicles with BTN synthesis and transport using Light Microscope (LM) immunocytochemistry. Using an antibody raised in rabbits against a mouse BTN peptide on sections of perfused sheep or goat mammary gland, we obtained an identical supranuclear localization to that produced by a rabbit anti-casein antibody (Uejyo et al. 2015). Casein is generally accepted to be processed in the Golgi and secreted via the Golgi vesicles (Beery et al. 1971; Linzell and Peaker 1971; Wooding 1977; Mather 2011). Other BTN antibodies sometimes show indications of such a localization but have never showed such reproducibility or uniformity. Materials & Methods Electron.