Here’s what various bosses look like animated with their sprite sheet replaced with Isaac’s sprite sheet. (Click on each GIF for captions)

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@thebrothingofisaac
Here’s what various bosses look like animated with their sprite sheet replaced with Isaac’s sprite sheet. (Click on each GIF for captions)

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In both the original version of The Binding of Isaac and its expansion Wrath of the Lamb, exhausting all of the items from an item pool would result in the game generating an invalid placeholder item that does nothing when collected. It rapidly flickers through every item sprite in the game and is simply called Undefined. This was later officially added in Rebirth as an actual active item complete with a glitched appearance as a direct reference to the bug found in the original game.
A very crude to-do list, made by a programmer during Afterbirth’s development.
Most floor themes include an extra instrumental layer that plays at the same time as the main floor theme when the player enters a room with many enemies in it. Here’s what the instrumental layer from Duress (the theme that plays in Sheol) sounds like without the accompanying music.
A piece of fan art made for Edmund Mcmillen that he posted to his personal blog.

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If the player manages to reach Delirium by either cheating or using the debug console and hasn’t fought a single boss on the run, Delirium will never transform.
Various sketches and pieces of concept art for the original Binding of Isaac made and posted by Edmund Mcmillen for a limited edition zine.
Left: One of the first attacking animations done for Ultra Greed during afterbirth’s development, posted by Tyrone Rodriguez.
Right: Ultra Greed’s final attacking animation, recorded from Aferbirth+’s animation editor.
The audio that plays when the player either uses The Gamekid or My Little Unicorn from the original Binding Of Isaac.
Concept art for the Meatball enemy, who was originally intended to appear in Rebirth but was scraped until it was later added in the Afterbirth expansion.

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Various screenshots from The Binding of Isaac: Rebirth’s release date trailer, which was also removed from Steam’s store page.
All of the large book sprites that appear on screen briefly when the player uses their respective items.
Top: A screenshot taken from the the starting room in the final version of The Binding of Isaac: Rebirth.
Bottom: The very first screenshot taken from a very early alpha build of the game. Note the lack of a HUD and the reflection in Isaac’s eyes being mirrored.
For some reason, the loading screen used in the original Binding of Isaac features two Scolex’s, two Pestilence’s, and four Larry Jr’s.
Every single boss Delirium can transform into, with the exception of hush’s second form, which remains unused in the game’s files.

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Most floor themes include an extra instrumental layer that plays at the same time as the main floor theme when the player enters a room with many enemies in it. Here’s what the instrumental layer from Sodden Hollows (the theme that plays in The Caves) sounds like without the accompanying music.
By going into the game’s options.ini file, one can change how far out the camera zooms both in game and in the menus. Here’s what the game looks like when the setting “MaxRender=99″ is changed to “MaxRender=1.”